Three-Dimensional In Vitro Staphylococcus aureus Abscess Communities Display Antibiotic Tolerance and Protection from Neutrophil Clearance

Marloes I Hofstee; Martijn Riool; Igors Terjajevs; Keith Thompson; Martin J Stoddart; R Geoff Richards; Sebastian A J Zaat; T Fintan Moriarty

doi:10.1128/IAI.00293-20

. 2020 Oct 19;88(11):e00293-20. doi: 10.1128/IAI.00293-20

Three-Dimensional In Vitro Staphylococcus aureus Abscess Communities Display Antibiotic Tolerance and Protection from Neutrophil Clearance

Marloes I Hofstee ^a,^b, Martijn Riool ^b, Igors Terjajevs ^a, Keith Thompson ^a, Martin J Stoddart ^a, R Geoff Richards ^a, Sebastian A J Zaat ^b, T Fintan Moriarty ^a,^✉

Editor: Nancy E Freitag^c

PMCID: PMC7573441 PMID: 32817328

KEYWORDS: Staphylococcus aureus, staphylococcal abscess communities, 3-dimensional, in vitro model, fibrin pseudocapsule, bacterium-host cell interactions, antibiotic tolerance, fibrin pseudocapsule

ABSTRACT

Staphylococcus aureus is a prominent human pathogen in bone and soft-tissue infections. Pathophysiology involves abscess formation, which consists of central staphylococcal abscess communities (SACs), surrounded by a fibrin pseudocapsule and infiltrating immune cells. Protection against the ingress of immune cells such as neutrophils, or tolerance to antibiotics, remains largely unknown for SACs and is limited by the lack of availability of in vitro models. We describe a three-dimensional in vitro model of SACs grown in a human plasma-supplemented collagen gel. The in vitro SACs reached their maximum size by 24 h and elaborated a fibrin pseudocapsule, as confirmed by electron and immunofluorescence microscopy. The in vitro SACs tolerated 100× the MIC of gentamicin alone and in combination with rifampin, while planktonic controls and mechanically dispersed SACs were efficiently killed. To simulate a host response, SACs were exposed to differentiated PLB-985 neutrophil-like (dPLB) cells and to primary human neutrophils at an early stage of SAC formation or after maturation at 24 h. Both cell types were unable to clear mature in vitro SACs, but dPLB cells prevented SAC growth upon early exposure before pseudocapsule maturation. Neutrophil exposure after plasmin pretreatment of the SACs resulted in a significant decrease in the number of bacteria within the SACs. The in vitro SAC model mimics key in vivo features, offers a new tool to study host-pathogen interactions and drug efficacy assessment, and has revealed the functionality of the S. aureus pseudocapsule in protecting the bacteria from host phagocytic responses and antibiotics.

INTRODUCTION

Staphylococcus aureus is a Gram-positive commensal bacterium that colonizes human nostrils and skin in approximately 20 to 40% of the world's population (1 –3). In contrast, S. aureus can also be an opportunistic pathogen causing skin and soft-tissue infections (SSTI), osteomyelitis, and orthopedic device-related infections (ODRI) (4), among others. Infections caused by S. aureus can be life-threatening due to bacteremia, metastatic infection, and septicemia (5).

Treatment of S. aureus infections can be challenging due to increasing antibiotic resistance and high treatment failure rates. For example, up to 20% of patients with SSTI (6) and chronic osteomyelitis patients (7) have relapsing infection, indicating the need for new, more effective treatment strategies.

S. aureus can cause abscess formation in tissue, as reported for SSTI (8), osteomyelitis (9), and endocarditis (10). These abscesses have been described to contain staphylococcal abscess communities (SACs), which are a dense population of S. aureus that envelope themselves with a pseudocapsule consisting of fibrin deposits (11 –13). The SAC is also surrounded by immune cells, in particular, neutrophils (11 –13). It has been proposed that S. aureus forms a SAC as a mechanism to escape host defenses (14 –16) and that the fibrin pseudocapsule enclosing the staphylococci function as a protective barrier, keeping immune cells at bay. However, no direct in vitro or in vivo evidence for this hypothesis has been presented so far. The SAC and immune cells become encapsulated by fibrous tissue, resulting in an abscess structure formed by the host in an attempt to at least contain the infection (11 –13). However, encapsulation by the host ultimately does not resolve the infection, since SACs remain viable over long periods in vivo (17, 18). This possibly explains, at least in part, the high rate of recurrence of infections in patients. Furthermore, antibiotic tolerance within a SAC may also contribute to recurrence of infection, but this has not yet been studied.

Due to coevolution with humans, S. aureus has numerous human-specific virulence-associated factors; therefore, animal models may not fully recapitulate these interactions (19 –21). Although active in murine plasma (17), staphylothrombin, which can convert fibrinogen into fibrin, displayed higher activity with human prothrombin than murine prothrombin (21), for example. This is of particular importance, since the pseudocapsule around the SAC contains fibrin (11 –13). Human-specific activity has also been proven for staphylokinase, the enzyme responsible for conversion of plasminogen into the active protease plasmin, which degrades fibrin (20). Therefore, working with human cells and factors appears crucial to effectively study these host-pathogen interactions for S. aureus in the context of human infection, and they cannot be adequately assessed using current preclinical models.

To address this potential limitation, numerous in vitro systems incorporating human cells in coculture with bacteria have been described (22 –24). In other infectious disease fields, such as intestinal or wound infections, the use of three-dimensional (3D) in vitro models is emerging (25, 26). These models contain human cells that may self-organize into in vivo tissue-like structures and are easily inoculated with pathogenic bacteria (26 –28). To date, only one study has described an in vitro S. aureus microcolony model to study S. aureus microcolony-neutrophil interactions (29). Although a promising approach and a valuable starting point, this study used an immature microcolony model, not a SAC model, which was exposed for short time periods to murine rather than human cells, and antibiotic tolerance of S. aureus in this model was not studied.

The aim of our study was to develop a 3D in vitro SAC model that recapitulates key in vivo characteristics, such as fibrin pseudocapsule formation, possible tolerance for antibiotics, and protection against human neutrophil clearance. We investigate the physical characteristics of the in vitro SAC model, possible tolerance of in vitro SACs to antibiotic treatment, and the efficacy of human neutrophils to eradicate staphylococci residing within SACs in the presence or absence of an intact fibrin pseudocapsule.

RESULTS

Morphological characteristics of the 3D in vitro SAC model.

S. aureus seeded at low density within a human plasma-supplemented collagen gel multiplied and formed an in vitro SAC (Fig. 1B). After 24 h of culture, these in vitro SACs consisted of a dense population of S. aureus within a pseudocapsule (Fig. 1B). The pseudocapsule contained a denser inner matrix and a more diffuse outer matrix (Fig. 1C and D), and both the inner and outer pseudocapsule layers were positively stained for fibrin (Fig. 1E).

Growth of in vitro SACs.

Growth of the in vitro SACs was assessed by CFU analysis (all SACs per sample) and imaging (representative SACs) over 72 h (Fig. 2). Staphylococci within the in vitro SAC model grew exponentially until 16 h, after which a plateau phase was reached (Fig. 2A). At 4 h, small S. aureus aggregates or immature SACs were observed (Fig. 2B, arrow). Pseudocapsules were present after 8 h of growth. At 16 h, the bacteria were surrounded by a clearly visible pseudocapsule around the SACs. After 24 h, the pseudocapsule around SACs remained clearly visible. By 48 h, satellite colonies were observed in some cases (Fig. 2B, arrowheads), and in other cases the SAC had lost its structural integrity. Moreover, at this time point the pseudocapsule started to degrade, and by 72 h the pseudocapsule was absent.

SAC formation by multiple S. aureus strains and clinical isolates.

To ensure that in vitro SAC formation was not specific for the tested S. aureus JAR 06.01.31 isolate, the model was repeated with multiple laboratory strains and clinical isolates of S. aureus and with a strain of S. epidermidis. Although there was some variation in the degree of pseudocapsule formation and the integrity of the pseudocapsules, all S. aureus strains and isolates formed in vitro SACs (Fig. 3A to J; images were sorted based on pseudocapsule size), which contained comparable numbers of bacteria at 24 h (Fig. 3L). More specifically, S. aureus Newman, S. aureus JAR 06.01.31, S. aureus ATCC 49230, methicillin-resistant S. aureus (MRSA) isolate Mu 81, and methicillin-susceptible S. aureus (MSSA) isolate Mu 30 had comparable pseudocapsules that were clearly observable (Fig. 3A to E). In contrast, S. aureus RN4220, MSSA Mu 102, and MRSA Mu 100 did not have a visible outer pseudocapsule (Fig. 3H to J), and MSSA Mu 8 and MRSA Mu 16 had an outer pseudocapsule that was different in appearance from those of the other in vitro SACs and appeared to be located at an increased distance (Fig. 3F and G). S. epidermidis 0-47 formed markedly smaller aggregates with no visible pseudocapsule and with fewer CFU per sample than S. aureus in vitro SACs (Fig. 3K and L).

FIG 3 — *In vitro* SAC formation by different *S. aureus* laboratory strains and clinical isolates and an *S. epidermidis* strain. Representative images of SACs from different *S. aureus* laboratory strains and clinical isolates and *S. epidermidis* strain O-47 at 24 h, sorted by pseudocapsule appearance. (A) *S. aureus* Newman. (B) *S. aureus* JAR 06.01.31. (C) *S. aureus* ATCC 49230. (D) MRSA Mu 81. (E) MSSA Mu 30. (F) MSSA Mu 8. (G) MRSA Mu 16. (H) *S. aureus* RN4220. (I) MSSA Mu 102. (J) MRSA Mu 100. (K) *S. epidermidis* O-47. Variation in pseudocapsule size, shape, and presence can be observed. Scale bar, 100 μm. (L) Quantitative culture after 24 h of growth.

Antibiotic tolerance of in vitro SACs.

Antibiotic susceptibility of the early and mature in vitro SACs, planktonic cultures, and mechanically dispersed SACs was determined. Immature in vitro SACs (4 h) were effectively killed after treatment with 100× the MIC of gentamicin for 3 h (P < 0.0001 versus untreated immature SACs) to a level comparable to that of the planktonic culture (P = 0.0003 versus untreated planktonic cultures) (Fig. 4A). In marked contrast, mature in vitro SACs (24 h) effectively tolerated exposure to 100× the MIC of gentamicin for 3 h (Fig. 4B) and for 24 h (Fig. 4C) and had bacterial numbers similar to those of untreated controls (P = 0.1820 and P = 0.6518, respectively), whereas the log-phase planktonic culture was highly susceptible at both times (P < 0.0001 in both cases). Stationary-phase bacteria were less susceptible to gentamicin treatment, but their survival was still significantly lower than that of nontreated controls (P = 0.0079). Numbers of CFU from mature SACs, both plasmin pretreated and treated with 100× the MIC of gentamicin, were not significantly different from those of nontreated controls (P = 0.4542). When mature SACs were dispersed and subsequently treated with 100× the MIC of gentamicin for 24 h, the bacteria were effectively killed by gentamicin, and samples contained significantly fewer bacteria than untreated dispersed SACs (P < 0.0001) (Fig. 4C).

Viability of the in vitro SACs after 24 h of exposure to gentamicin was assessed with Live/Dead staining (Fig. 4D, images 1 to 3). Untreated mature in vitro SACs lost their structural integrity more than antibiotic-treated mature in vitro SACs, characterized by an irregular outer edge (Fig. 4D, image 1). However, despite gentamicin treatment, dead bacteria did not predominate in mature SACs under both nontreated and gentamicin-treated conditions (Fig. 4D, images 1 and 2, propidium iodide [PI; red]). Mature SACs that were both plasmin pretreated and gentamicin treated contained PI-stained bacteria, indicative for dead cells, in the outer rim of the structure (Fig. 4D, image 3). Exposure to fluorescently labeled gentamicin showed that only a minimal amount of gentamicin reached bacteria in the outer rim of a mature SAC, whereas more gentamicin was present in the outer rim of a plasmin-pretreated mature SAC, being in line with the Live/Dead staining results (Fig. 4D, images 4 and 5, respectively). The isopropanol-treated SAC contained PI-stained cells, confirmatory for dead bacteria (Fig. 4D, image 6, PI [red]). The planktonic log-phase bacteria treated for 24 h with 100× the MIC of gentamicin appeared intact, but the Live/Dead staining showed them to be PI positive, indicating that these bacteria were dead, which is in line with the above-mentioned quantitative cultures (Fig. 4D, image 7). In addition, a large amount of cell debris was present in 24-h antibiotic-treated planktonic samples, indicating cell death and loss of integrity (data not shown). Similar results, both for quantitative cultures and Live/Dead stainings, were observed when SACs were treated with the combination of gentamicin and rifampin (see Fig. S1 in the supplemental material).

Interactions of dPLB cells with in vitro SACs.

To evaluate host cell interactions with in vitro SACs, differentiated neutrophil-like differentiated PLB-985 neutrophil-like (dPLB) cells were added to planktonic bacteria (0 h), immature SACs (4 h), and mature SACs with or without a 2-h plasmin pretreatment (24 h) (Fig. 5A). When added to planktonic bacteria and immature SACs, the dPLB cells effectively prevented bacterial growth into a mature SAC and actually reduced the numbers of bacteria relative to the numbers at the start of incubation (P < 0.0001 and P = 0.0002, respectively). However, the addition of dPLB cells to mature SACs did not significantly decrease bacterial numbers compared to the initial number of CFU (P = 0.1102). Plasmin pretreatment of mature SAC was performed to degrade the pseudocapsule. Mature SACs pretreated with plasmin for 2 h, prior to addition of dPLB cells for 24 h, contained fewer bacteria than non-plasmin-pretreated equivalents (P = 0.0333) and fewer bacteria than that at the start of incubation (P = 0.0002).

dPLB cells localized near the mature SAC (Fig. 5B), around the inner fibrin pseudocapsule (Fig. 5C, black arrow), but the dPLB cells (Fig. 5C, red arrowheads) did not penetrate it. The dPLB cells around the SAC were a heterogeneous population of live and dead cells, while the bacteria of the SAC were alive (Fig. 5D). Fibrin degradation of a plasmin-pretreated mature SAC occurred on a variable basis; the SAC kept its round shape or the SAC fell apart. dPLB cells invaded the SAC that was pretreated with plasmin (Fig. 5E) and were found to have internalized bacteria (Fig. 5F), although dPLB cells within the SAC were PI stained (Fig. 5G).

Interactions of primary human neutrophils with in vitro SACs.

To maximally mimic the in vivo human situation, mature SACs with or without 2 h of plasmin pretreatment were exposed for 6 or 24 h to primary human neutrophils (Fig. 6A). Upon primary human neutrophil exposure for 6 h, plasmin-pretreated mature SACs had lower CFU counts per sample than non-plasmin-pretreated mature SACs (P = 0.0272) (Fig. 6A) and, in contrast to non-plasmin-pretreated mature SACs (Fig. 6B), were observed to contain neutrophils (Fig. 6D). However, plasmin-pretreated mature SACs and non-pretreated mature SACs exposed to primary human neutrophils for 24 h had similar CFU counts per sample (P = 0.1711) (Fig. 6A). Nevertheless, microscopic differences were observed. Non-plasmin-pretreated mature SACs exposed to primary human neutrophils for 24 h appeared to contain fewer neutrophils within the SAC bacterial community (Fig. 6C) than plasmin-pretreated mature SACs (Fig. 6E). The many primary human neutrophils that invaded the plasmin-pretreated mature SACs during the 24-h coincubation stained positive for PI, indicating dead cells (Fig. 6E).

FIG 6 — Interaction between primary human neutrophils and a SAC. (A) Numbers of *S. aureus* JAR 06.01.31 CFU cultured at 6 or 24 h after the addition of primary human neutrophils to mature *in vitro* SACs that were nontreated or plasmin pretreated. Holm-Sidak’s multiple-comparison test was used. Data are mean ± SD numbers of CFU per sample and are from three independent experiments. (B and C) Confocal images of a mature *in vitro* SAC exposed for 6 (B) or 24 (C) h to primary human neutrophils. (D and E) Invasion of a plasmin-pretreated mature SAC by primary human neutrophils after 6 (D) or 24 (E) h of exposure. These SACs were treated with plasmin for 2 h and subsequently exposed for 6 or 24 h to primary human neutrophils. Samples for confocal imaging were stained with propidium iodide (PI) and Syto9. Green, both bacteria and primary human neutrophils stained with nucleic acid dye Syto9, indicating all cells. Red, PI-stained cells, indicating dead cells. Scale bars, 100 μm (B and C), 50 μm (D), and 100 μm (E). N.S., *P >* 0.05; *, *P <* 0.05.

Morphological comparison of in vivo and in vitro SACs.

In vivo and in vitro SACs were stained with a Picro-Mallory trichrome stain to compare their characteristics. In vivo samples consisted of murine femoral bone infected with S. aureus for 21 days. These samples contained abscesses with SACs in bone marrow. In vivo, the inner part of the SAC, which contained the bacteria, stained blue, indicating the presence of connective tissue and/or mucopolysaccharides (Fig. 7A). The in vivo SACs were surrounded by a fibrin pseudocapsule, as shown by the magenta stain and by many granulocytic cells. The 3D in vitro SAC model had a similar staining pattern (Fig. 7B). The bacterial core of the SAC stained blue for connective tissue and/or mucopolysaccharides, the pseudocapsule stained magenta for fibrin, and dPLB cells were present around the SAC.

FIG 7 — Morphological comparison between *in vivo* SACs in mice and *in vitro* SACs. Infected *in vivo* bone samples were obtained from C57BL/6 mice that received a double osteotomy of the femur, an inoculum of 4 log₁₀ CFU *S. aureus* JAR 06.01.31, and stabilization with a titanium 6-hole MouseFix locking plate. Paraffin-embedded *in vivo* (A) and *in vitro* (B) samples were stained with a Picro-Mallory trichrome stain, where connective tissue and mucopolysaccharides stain blue, muscles and erythrocytes stain yellow, and fibrin stains magenta. Nuclei of cells are stained purple/brown. Scale bars, 20 μm.

DISCUSSION

S. aureus is a key pathogen in a number of clinically important infections. In addition to biofilm formation (30) and intracellular survival (31), one of the key features of S. aureus infection is SACs and the abscesses that form around them (11, 17, 18, 32). Considering the importance of SAC formation to initiation and persistence of infection, and the specificity of many S. aureus virulence factors for human targets, it is crucial to have adequate models to enable mechanistic studies on SAC formation and persistence. In this study, we developed a 3D in vitro SAC model that resembles key, known in vivo characteristics of SACs, such as a dense core of bacteria surrounded by a fibrin pseudocapsule. These basic features were observed with a series of S. aureus clinical isolates and laboratory strains to various degrees. In vivo, SACs are also surrounded by immune cells, primarily neutrophils, with relatively few monocytes and lymphocytes (11, 13, 16, 17). Following the incorporation of human neutrophils into our 3D in vitro SAC model, neutrophils accumulated at the periphery of the SAC, similar to what has been observed in vivo. This is the first time a comparison between in vitro and in vivo SACs has been reported, and it illustrates the potential utility of the model for further investigations into SAC establishment and persistence or interventions targeting eradication.

A key feature of S. aureus infection is the ability to develop a fibrin matrix at the periphery of the SAC. The in vitro SAC model contained an inner and outer fibrin pseudocapsule. It is known that S. aureus secretes two coagulant proteins, coagulase and von Willebrand factor-binding protein (vWbp). Coagulase acts in relative close proximity to the bacterial cell, whereas vWbp diffuses away from the bacteria, as shown in vivo (17), and is active at an increased distance in vitro, as shown in previously studied S. aureus microcolonies (29). Both coagulase and vWbp are capable of binding prothrombin to form catalytically active thrombin, which will subsequently convert fibrinogen into fibrin (16, 33, 34). The inner pseudocapsule observed around the in vitro SACs may result from coagulase activity, and outer pseudocapsules may result from vWbp activity.

The interstrain/isolate comparison performed in our study demonstrated differences in the diameter of the outer pseudocapsule diameter. Furthermore, some S. aureus cells formed SACs with a more distant outer pseudocapsule, not directly in contact with the inner pseudocapsule. This may be a result of vWbp activity, since S. aureus lacking vWbp is unable to form an outer fibrin meshwork (29). Interestingly, S. aureus RN4220, MSSA Mu102, and MRSA Mu100 appeared to lack pseudocapsules. S. aureus RN4220 has a mutation in its clfA gene (35) encoding clumping factor A (ClfA), a surface protein able to bind fibrinogen. Given this mutation, this strain may have a reduced affinity for fibrinogen (35), possibly causing its SACs to be devoid of an outer pseudocapsule. Our results suggest that MSSA Mu102 and MRSA Mu100 isolates also have a reduced ability to bind fibrinogen, although this remains to be determined.

The pseudocapsule was degraded when the in vitro SAC model was in culture for longer than 48 h. A potential explanation is that this happened via activity of staphylokinase, which is regulated by the agr system (36), as previously described (29). Staphylokinase is a plasminogen activator causing plasmin formation and subsequent fibrin degradation by this plasmin (20). In vitro it has been demonstrated that S. aureus secretes staphylokinase after entering the stationary phase of growth (37), which matches the time course of the disappearance of the pseudocapsule in our in vitro SAC model. In vivo, SACs retain their pseudocapsule for a longer period, as shown for day 21 in mice in Fig. 7A. The SACs may act to maintain protection against host immune cells but may also be a response to antimicrobial therapy by forcing bacteria into a slow-growing/dormant state due to low nutrient levels within a SAC, as shown for biofilm (38). The dispersal of the bacteria from the SAC by breakdown of the pseudocapsule, as seen in this model, may reflect a mechanism for S. aureus to disseminate, as is known to occur for abscesses (39), and to form new SACs elsewhere. The precise mechanisms involved in pseudocapsule degradation, conditions favoring retention of the pseudocapsule remain to be elucidated, and the presented model is well placed to enable such investigations.

A feature of interest is the lack of activity of antibiotics against S. aureus infection, as often observed in vivo (40), but which has not been specifically reported for SACs. The in vitro SAC model tolerated 100× the MIC of gentamicin and also the combination of gentamicin and rifampin. Antibiotic tolerance of fibrin containing S. aureus aggregates in vitro has been previously described (41), and fibrin presence itself may be involved in tolerance against antibiotics (32, 42). Similar to in vitro SACs, S. aureus biofilms within a matrix are tolerant to a wide range of antibiotics and antimicrobials (38, 43), as well as when the biofilm is covered with a fibrin-containing matrix (44). Bacteria of dispersed biofilm or biofilm that does not contain a matrix lose their antimicrobial-tolerant phenotype, which may result from the loss of protection by the matrix (38, 45, 46) and no longer being in a tightly packed structure (47). By exposing SACs to fluorescently labeled gentamicin, we could determine that, indeed, the fibrin pseudocapsule and the tightly packed nature of a SAC play a role in the antibiotic tolerance of in vitro SACs. We observed only minimal amounts of gentamicin within a mature SAC, whereas plasmin-pretreated mature SACs without a fibrin pseudocapsule contained gentamicin in the outer rim of the SAC, and bacteria stained positive for the cell death marker PI. When the SACs were dispersed in our study, the bacteria became susceptible to the antibiotics and were killed. It cannot be ruled out that the growth phase of the bacteria within a SAC influences this phenomenon. It may be that upon dispersal of the SAC-containing collagen gel, the bacteria regain metabolic activity and, therefore, lose their antibiotic tolerance.

Another characteristic in vivo feature of S. aureus infection that our in vitro model sought to recapitulate and examine was the proposed protective effect of the pseudocapsule around a SAC against immune cells, particularly neutrophils (14, 16, 32). Experimental in vivo studies have shown that mice with renal S. aureus infection had SACs that were surrounded by an inner zone of dead neutrophils, an outer zone of viable neutrophils, and finally a rim of necrotic neutrophils in their kidneys (14, 16, 39). We show that dPLB cells as well as primary human neutrophils migrate through the collagen gel toward the in vitro SACs. However, after 6 or 24 h, primary human neutrophils or dPLB cells, respectively, do not penetrate the fibrin pseudocapsule to enter established SACs, and approximately half of the dPLB cell population and even less of the primary human neutrophil population appeared dead at the periphery of the SAC. The physical appearances of the in vitro and in vivo SACs shown in Fig. 7 suggest that the margin of the SAC, demarcated by the pseudocapsule, is a key interface protecting the bacteria from host defenses, which is in line with earlier reported in vitro results (29). Besides the pseudocapsule being a mechanical barrier to prevent neutrophil invasion into a SAC, the fibrin pseudocapsule may reduce neutrophil activity as well (21). Further investigation of host-pathogen interactions at this interface is of significant interest.

Even after the degradation of the fibrin pseudocapsule to facilitate neutrophil access, in vitro SACs were not completely cleared. The SACs have an approximately 11,000-fold larger volume than a neutrophil, contain 8 log₁₀ CFU, and are approximately 200 μm in diameter, compared to an average diameter of approximately 9 μm for neutrophils (48). Although the neutrophils attempt to clear the bacteria, they are significantly disadvantaged in the in vitro SAC model. However, the dPLB cells were able to contain the infection at earlier time points, when the bacterial numbers were still low and SACs did not have a pseudocapsule yet. Primary human neutrophils decreased bacterial numbers of plasmin-pretreated SACs within 6 h after adding these phagocytes, whereas bacterial clearance by dPLB cells took much longer and was only recorded at 24 h after their addition. This difference may be explained by the fact that primary human neutrophils have stronger responsiveness toward chemotactic stimuli than dPLB cells (49). This way, they will reach the bacteria earlier and mediate their clearance more efficiently.

Surprisingly, we observed many dead neutrophils within plasmin-pretreated SACs. It has been reported that S. aureus aggregates in the presence of fibrinogen upregulate quorum sensing-dependent virulence modulated by the agr system (50). The agr system controls many toxins, for example, phenol-soluble modulins and pore-forming toxins, that can affect neutrophils (51). It may be that even in the absence of fibrin (plasmin pretreatment), SACs still produce phenol soluble modulins, which both attract and kill neutrophils (52), or other pore-forming S. aureus toxins, such as leukotoxins, that effectively kill neutrophils by cell lysis (53); therefore, neutrophils within plasmin-pretreated SACs were easily killed. The dead neutrophils may provide new nutrients for the bacteria (54), allowing the SACs to grow. This will likely easily even out any possible decrease in bacterial numbers due to neutrophil action, and this would clarify why the decrease in bacterial numbers of plasmin-pretreated SACs measured after 6 h of exposure with primary human neutrophils was not observed anymore after 24 h of exposure to these phagocytes.

In the future, the in vitro SAC model may be used to investigate other basic interactions between S. aureus and host cells to identify new drug targets for S. aureus infections. Furthermore, the model may be used as a prescreening tool for a wide variety of new S. aureus infection treatment strategies to reduce the number of animal studies required. An approach for a new S. aureus infection treatment could be to target the fibrin pseudocapsule either directly or indirectly. Directly, a fibrinolytic compound may be used or fibrin(ogen) binding by the bacterium may be inhibited, and, indirectly, the conversion of fibrinogen to fibrin by coagulase and vWbp may be prevented. Using the model, the pathogenesis of persistent S. aureus SAC infections not responding to antibiotic therapy will become better understood, and novel targets will help prevent and treat these dreaded infections.

Conclusions.

In this study, we developed a 3D in vitro SAC model to study antibiotic tolerance and pathogen-host cell interactions while recapitulating features of the in vivo situation. In vitro SACs were covered by a fibrin pseudocapsule, human neutrophils were unable to reach staphylococci within in vitro SACs, and in vitro SACs displayed antibiotic tolerance. We also demonstrated that the fibrin pseudocapsule is a key feature of the in vitro SACs, first reducing susceptibility to human neutrophils and, second, together with the tightly packed nature of a SAC, preventing antibiotics from diffusing into a SAC, causing antibiotic tolerance. Taken together, the in vitro SAC model mimics key in vivo features, offers a new tool to study host-pathogen interactions and drug efficacy assessment, and has revealed the functionality of the S. aureus pseudocapsule in protecting the bacteria from host phagocytic responses and antibiotics.

MATERIALS AND METHODS

Bacteria.

S. aureus JAR 06.01.31 (number 890; the Culture Collection of Switzerland [CCOS], Wädenswill, Switzerland), obtained from a patient with a periprosthetic joint infection (55), was used for the in vitro SAC model. The MIC of gentamicin and rifampin for this isolate is 1 μg/ml and 0.004 μg/ml, respectively (measured by Synlab, Luzern, Switzerland).

In addition, a panel of clinical isolates and laboratory strains was used to compare SAC forming ability and pseudocapsule formation among staphylococci. The laboratory strains used were S. aureus Newman (CCOS number 199), S. aureus ATCC 49230 (American Type Culture Collection [ATCC], Manassas, United States), and S. aureus RN4220 (ATCC 35556). The clinical isolates used were collected at the Trauma Center Murnau (Germany) from patients with ODRI and included methicillin-susceptible S. aureus (MSSA) isolates Mu 8, Mu 30, and Mu 102 and methicillin-resistant S. aureus (MRSA) isolates Mu 16, Mu 81, and Mu 100. S. epidermidis strain O-47, originally isolated from a patient at the Institute Für Medizinische Mikrobiologie und Hygiene, University of Cologne (Germany) (56), was also included as a coagulase-negative comparator strain.

Bacterial stock solutions were stored in tryptic soy broth (TSB; Oxoid, Basel, Switzerland) containing 20% (vol/vol) glycerol at –20°C.

General SAC methodology.

Bacteria were recovered from frozen stocks and were grown overnight on tryptic soy agar (TSA; Oxoid) plates at 37°C. Colonies were then resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen, Basel, Switzerland) to an optical density at 620 nm (OD₆₂₀) of 0.7 ± 0.05 and then diluted 1:200,000 to obtain a cell suspension containing approximately 14 CFU in 25 μl.

A schematic overview of the different components of the in vitro SAC model is shown in Fig. 1A. Collagen gel was prepared from rat collagen I solution (Gibco, Basel, Switzerland) by following the manufacturer's instructions to obtain a final concentration of 1.78 mg/ml in 1× RPMI 1640 medium (pH 7.4). Fifty microliters of collagen gel was added per well of 24-well Transwell systems (polyester membrane with a porosity of 0.4 μm; Corning Life Sciences B.V., Amsterdam, the Netherlands), and 80 μl collagen gel was added when using 48-well plates (TPP, Trasadingen, Switzerland). After polymerization for 1 h at 37°C in a humidified incubator, 25 μl of bacterial suspension (described above) was pipetted on top of the collagen gel. After 1 h at 37°C, an additional collagen gel layer of 25 μl (24-well Transwell) or 40 μl (48-well) was pipetted on top of the bacterial solution, and this upper gel layer was allowed to polymerize for 30 min at 37°C in a humidified incubator. After 30 min, 200 μl pooled human plasma (Regional Blood Donation Service SRK Graubünden, Chur, Switzerland) was added to the well, and after 5 h an additional 200 μl pooled human plasma was provided.

To quantify bacterial numbers, samples were transferred to self-standing 50-ml tubes (TPP) containing 1 mm zirconium oxide beads (Next Advance, New York, NY) and 250 μl phosphate-buffered saline (PBS; Gibco). Samples were homogenized with a Bullet Blender (Next Advance) for 3 min (speed 10) and sonicated (Bandelin Electronic, Berlin, Germany) for 3 min at 35 kHz. The sample solutions were 10-fold serially diluted, pipetted on TSA plates, and incubated overnight at 37°C to determine the number of CFU.

To prepare sections of the in vitro SACs for microscopy, samples were fixed with McDowell’s fixative, dehydrated, and embedded in paraffin.

To measure the growth of SACs over 72 h, SACs were cultivated for 0, 4, 8, 16, 24, 48, and 72 h, and the total numbers of CFU were determined at each time point. In the cell interaction studies, neutrophils were added at 24 h in 60 μl collagen gel overlaid with 200 μl RPMI 1640 medium, as described below. To replicate these conditions in the bacterium-only experiments, an additional 60 μl collagen gel (prepared as described above) and 200 μl RPMI 1640 medium were added to the in vitro SAC model after 24 h.

Morphological analysis, antibiotic tolerance, and host cell-bacterium interaction experiments were performed with 24-well Transwell systems. The growth experiments were performed in a 48-well plate, since samples did not have to be imaged with a confocal microscope, removed intact from the well or exposed to treatments. All growth, antibiotic tolerance, and host cell-bacterium interaction experiments were repeated three times and with a minimum of triplicate samples per repetition. Quantitative cultures were performed of the total in vitro model, including all SACs in each sample.

Host cells.

The human acute myeloid leukemia cell line PLB-985 (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany) was cultured in RPMI 1640 medium containing l-glutamine, 25 mM HEPES, 10% (vol/vol) fetal bovine serum (FBS; Sigma-Aldrich, Buchs, Switzerland), and 1% (vol/vol) penicillin-streptomycin solution (Pen/Strep; Sigma-Aldrich) at 37°C and 5% CO₂ and was maintained at a cell density between 0.2 × 10⁶ and 1 × 10⁶ cells/ml. To differentiate the PLB-985 cells into a neutrophil-like phenotype (dPLB cells), 0.5 × 10⁵ cells/ml were cultured in RPMI 1640 medium containing 5% (vol/vol) FBS and 1.25% (vol/vol) dimethyl sulfoxide (Sigma-Aldrich) for 5 days (57), with a culture medium change on the third day (57).

For the isolation of primary human neutrophils, fresh blood was collected (using heparin as an anticoagulant) from healthy volunteers as part of the blood sampling for biomedical experiments (BACON) study protocol, version 1.8, 26 October 2018, of the Amsterdam UMC and in compliance with the Declaration of Helsinki. Blood was diluted 1:1 with Hanks’ balanced salt solution (HBSS; Sigma-Aldrich, Houten, the Netherlands), loaded onto 20 ml Lymphoprep (ELITechGroup, Zottegem, Belgium), and centrifuged at 1,200 × g for 30 min at room temperature (RT). The erythrocyte pellet containing granulocytes was then transferred to a new 50-ml tube, and erythrocytes were lysed with ice-cold erythrocyte lysis buffer (155 mM NH₄Cl [Sigma-Aldrich], 10 mM KHCO₃ [Merck KGaA, Darmstadt, Germany], 80 μM EDTA [Merck KGaA] in double-distilled water with a pH of 7.3). Nonlysed cells in lysis buffer were centrifuged at 400 × g for 10 min at 10°C. Subsequently, cells were resuspended in fresh lysis buffer, put on ice for 5 min, and further centrifuged to remove any remaining cell debris. Lastly, the cells were washed with ice-cold PBS and centrifuged at 400 × g for 10 min at 10°C, and the supernatant was removed, leaving a white cell pellet, which was resuspended in collagen gel. Flow-cytometric analysis showed that the final cell suspensions contained more than 98% granulocytes based on forward-scatter and side-scatter profiles, of which at least 96% were neutrophils, as determined with CD15 and CD16 stains.

Antibiotic tolerance of in vitro SACs.

For antibiotic tolerance testing of bacteria in SACs, PBS containing 100 μg/ml gentamicin sulfate (Roth AG, Arlesheim, Switzerland), or 100 μg/ml gentamicin in combination with 0.4 μg/ml rifampin (Roth AG), i.e., 100 times the MIC, was used. SACs were challenged after 4 h (immature SACs) or 24 h (mature SACs) of growth by applying 200 μl gentamicin-containing PBS solution on top of the gel, after removing the human plasma, and 600 μl into the well underlying the Transwell insert. Immature SACs were challenged for 3 h, whereas mature SACs were challenged for 3 h or 24 h. In several experiments, mature, 24-h-grown SACs were plasmin pretreated to degrade the fibrin pseudocapsule around SACs by first removing the human plasma from the SAC-containing gel and incubating samples for 2 h with plasmin (8 μg/ml; Sigma-Aldrich) diluted in PBS, 75 μl within the Transwell and 125 μl into the well underlying the Transwell insert. After the plasmin pretreatment, plasmin was removed and gentamicin-containing PBS was added to the samples as described before. For the combination of gentamicin and rifampin, non-pretreated or plasmin-pretreated mature SACs were challenged for 24 h with PBS containing gentamicin and rifampin as described above. To obtain dispersed mature SACs, the SACs were first homogenized and sonicated as described above. Dispersed mature SACs were treated for 24 h with either gentamicin alone or gentamicin plus rifampin at the above-mentioned concentrations.

Planktonic bacteria used as controls were grown to log phase or to stationary phase (24 h of incubation) in TSB. The same inoculum was used for planktonic bacteria and the in vitro SAC model. The planktonic bacteria were centrifuged at 1,223 × g for 5 min and the supernatant removed, and bacterial pellets were resuspended in 800 μl PBS in the presence or absence of gentamicin alone or in combination with rifampin at the above-mentioned concentrations. Planktonic bacteria were treated for 3 h or 24 h with the antibiotic solutions. For Live/Dead staining, planktonic bacteria were embedded in 10 μl collagen gel (1.78 mg/ml) after antibiotic treatment and 3 washes with PBS.

As a positive control for dead bacteria within a SAC, mature SACs were treated for 30 min with 70% isopropanol (Roth AG). The same application method and volumes as those for the antibiotics were used.

Interactions of dPLB cells or primary human neutrophils with in vitro SACs.

To assess SAC-neutrophil interactions, dPLB cells were added to SACs grown for 0, 4, or 24 h. Mature, 24-h grown SACs were either not treated or plasmin pretreated. Before addition of plasmin or dPLB cells, human plasma was aspired from the in vitro SACs. Plasmin (8 μg/ml in PBS) pretreatment was performed for 2 h (see the method described above) to degrade the fibrin pseudocapsule of mature SACs prior to the addition of the dPLB cells when indicated. After 2 h, the plasmin solution was removed. dPLB cells were mixed into a collagen gel on ice (1 × 10⁶ cells in 60 μl collagen gel/sample) and were added to nontreated or plasmin-pretreated in vitro SACs, forming an additional layer on top of the gel with the in vitro SACs. After the gel had polymerized for 30 min at 37°C with 5% CO₂, 200 μl RPMI was added on top of the dPLB cell-containing collagen gel layer, and samples were incubated for 48 h at 37°C with 5% CO₂.

Primary human neutrophils (1 × 10⁶ cells in 60 μl collagen gel/sample) were added to mature SACs with or without plasmin pretreatment in the same manner as that described above. After 6 or 24 h of incubation at 37°C with 5% CO₂, bacterial clearance by the primary human neutrophils was assessed by quantitative culture. Three independent experiments were performed using a different primary human neutrophil donor for each of the experiments.

In microscopic images where bacteria and human neutrophils were stained with the same dye, bacteria and dPLB cells or primary human neutrophils were distinguished from one another based on size.

In vivo samples.

Paraffin sections of S. aureus-infected murine femoral bone were obtained from a study approved by the ethical committee of the canton of Graubünden in Switzerland (TVB 2017_28). Inoculum preparation and the surgical procedure were performed as previously described (58). Briefly, female, specific-pathogen-free (SPF), 20- to 28-week-old C57BL/6 mice (Charles Rivers, Sulzfeld, Germany) received a double osteotomy of the femur, creating a 2-mm defect, with stabilization provided by a titanium 6-hole MouseFix locking plate (RISystems AG, Davos Platz, Switzerland). The bone marrow of the created 2-mm defect was inoculated with 4 log₁₀ CFU of S. aureus JAR 06.01.31 and placed back into the defect space. Animals were sacrificed after 21 days, femurs fixed in formalin and decalcified with ethylenediaminetetraacetic acid (EDTA) decalcification solution, implants removed, and the remaining bone and soft tissue embedded in paraffin.

Stainings.

The Live/Dead BacLight bacterial viability kit (Invitrogen) was used by following the manufacturer's instructions. Briefly, 500 μl of the staining solution, including Syto9 (all cells) and propidium iodide (PI; dead cells), was applied per sample. The membrane dye PKH26 (Sigma-Aldrich) was used to stain dPLB-985 cells (0.4 μl PKH26/1 × 10⁶ dPLB cells) by following the manufacturer's instructions and was combined with 200 μl of Syto9 staining solution to stain the SAC. Texas red-gentamicin (2 μg/sample; Ursa BioScience Llc., Bel Air, MD) was applied in 200 μl gentamicin-containing PBS on top of the collagen gel with SACs for 24 h. SACs were stained with 200 μl of Syto9 staining solution.

Sectioned paraffin-embedded in vitro and in vivo samples (5 μm thick) were postfixed with Bouin’s fluid (0.9% water-saturated picric acid, 9% formaldehyde, and 5% acetic acid) overnight at RT and then washed by rinsing sections in distilled water. Weigert’s iron hematoxylin (Sigma-Aldrich) was applied to stain nuclei, and samples were stained with a Picro-Mallory trichrome stain to stain fibrin by following a previously described protocol (59). Stained paraffin sections were dehydrated with three absolute ethanol steps, cleared 2 times in pure xylene for 5 min, and mounted with Eukitt mounting medium (Sigma-Aldrich).

The staining steps described below were performed with 200 μl of the indicated solutions unless stated otherwise. Samples were blocked with 1:20 diluted goat serum (Reactolab, S. A., Servion, Switzerland) in PBS with 1% Triton X (PBS-T; Sigma-Aldrich) for 1 h at RT. Blocking buffer was removed, and the primary rabbit anti-human fibrinogen antibody (1:200 dilution; Innovative Research, Novi, MI) was added to the samples and incubated for 1 h at RT. Subsequently, samples were washed 3 times with 500 μl PBS-T for 5 min. Goat anti-rabbit IgG (H+L) highly cross-adsorbed Alexa Fluor 568 (Invitrogen) was used as a secondary antibody (1:200 dilution), and samples were incubated for 30 min in the dark at RT, followed by 3 washes with 500 μl PBS-T for 5 min.

Microscopy.

Confocal laser scanning microscopy was performed using a Zeiss LSM 800 microscope or a Leica TCS SP8 X microscope. Phase contrast images were taken with the Zeiss Axio Vert.A1 microscope (Zeiss, Oberkochen, Germany). Image processing was performed with ZEN (blue edition) software (Zeiss).

Samples for transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were fixed with McDowell’s fixative and dehydrated with an ascending ethanol series. For TEM, LX112 resin embedding (Ladd Research, Willeston, VT) was used, after which the samples were sectioned and stained with 3.5% aqueous uranyl acetate solution for 3 to 4 min and, subsequently, 2% lead citrate solution for 1 to 2 min. Images were taken with an FEI Tecnai T12 TEM microscope. SEM samples were paraffin embedded, sectioned, deparaffinized, air dried, sputter coated with gold/palladium, and imaged with a Hitachi S-4700 field emission scanning electron microscope.

Statistical analysis.

Statistical analysis was performed with GraphPad Prism 8 (GraphPad Software, San Diego, CA). The normality of the log-transformed data was checked with a Shapiro-Wilk test and by visual inspection of the associated Q-Q plot. A Holm-Sidak`s multiple-comparison test was used to compare CFU counts of samples. P values of <0.05 were considered statistically significant.

Supplementary Material

Supplemental file 1

IAI.00293-20-s0001.pdf^{(868.3KB, pdf)}

ACKNOWLEDGMENTS

We acknowledge P. Furlong for SEM imaging and the Cellular Imaging Core Facility of the Amsterdam UMC, location AMC, for TEM imaging. We thank E.C. de Jong for her critical expertise and advice and F. M. J. Hafkamp for acquiring the primary human neutrophils.

This work was supported by AOTrauma as part of the Clinical Priority Program on Bone Infection.

Footnotes

Supplemental material is available online only.

REFERENCES

1.den Heijer CD, van Bijnen EM, Paget WJ, Pringle M, Goossens H, Bruggeman CA, Schellevis FG, Stobberingh EE, APRES Study Team. 2013. Prevalence and resistance of commensal Staphylococcus aureus, including meticillin-resistant S aureus, in nine European countries: a cross-sectional study. Lancet Infect Dis 13:409–415. doi: 10.1016/S1473-3099(13)70036-7. [DOI] [PubMed] [Google Scholar]
2.Kluytmans J, van Belkum A, Verbrugh H. 1997. Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clin Microbiol Rev 10:505–520. doi: 10.1128/CMR.10.3.505-520.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Morgenstern M, Erichsen C, Hackl S, Mily J, Militz M, Friederichs J, Hungerer S, Buhren V, Moriarty TF, Post V, Richards RG, Kates SL. 2016. Antibiotic resistance of commensal Staphylococcus aureus and coagulase-negative staphylococci in an international cohort of surgeons: a prospective point-prevalence study. PLoS One 11:e0148437. doi: 10.1371/journal.pone.0148437. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Tong SY, Davis JS, Eichenberger E, Holland TL, Fowler VG Jr. 2015. Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev 28:603–661. doi: 10.1128/CMR.00134-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Lowy FD. 1998. Staphylococcus aureus infections. N Engl J Med 339:520–532. doi: 10.1056/NEJM199808203390806. [DOI] [PubMed] [Google Scholar]
6.Sreeramoju P, Porbandarwalla NS, Arango J, Latham K, Dent DL, Stewart RM, Patterson JE. 2011. Recurrent skin and soft tissue infections due to methicillin-resistant Staphylococcus aureus requiring operative debridement. Am J Surg 201:216–220. doi: 10.1016/j.amjsurg.2009.12.024. [DOI] [PubMed] [Google Scholar]
7.Conterno LO, Turchi MD. 2013. Antibiotics for treating chronic osteomyelitis in adults. Cochrane Database Syst Rev 9:CD004439. doi: 10.1002/14651858.CD004439.pub3:CD004439. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Bae IG, Tonthat GT, Stryjewski ME, Rude TH, Reilly LF, Barriere SL, Genter FC, Corey GR, Fowler VG Jr. 2009. Presence of genes encoding the Panton-valentine leukocidin exotoxin is not the primary determinant of outcome in patients with complicated skin and skin structure infections due to methicillin-resistant Staphylococcus aureus: results of a multinational trial. J Clin Microbiol 47:3952–3957. doi: 10.1128/JCM.01643-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Ochsner PE, Hailemariam S. 2006. Histology of osteosynthesis associated bone infection. Injury 37:S49–S58. doi: 10.1016/j.injury.2006.04.009. [DOI] [PubMed] [Google Scholar]
10.Miro JM, Anguera I, Cabell CH, Chen AY, Stafford JA, Corey GR, Olaison L, Eykyn S, Hoen B, Abrutyn E, Raoult D, Bayer A, Fowler VG Jr, International Collaboration on Endocarditis Merged Database Study Group. 2005. Staphylococcus aureus native valve infective endocarditis: report of 566 episodes from the International Collaboration on Endocarditis Merged Database. Clin Infect Dis 41:507–514. doi: 10.1086/431979. [DOI] [PubMed] [Google Scholar]
11.Brandt SL, Putnam NE, Cassat JE, Serezani CH. 2018. Innate immunity to Staphylococcus aureus: evolving paradigms in soft tissue and invasive infections. J Immunol 200:3871–3880. doi: 10.4049/jimmunol.1701574. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Farnsworth CW, Schott EM, Benvie AM, Zukoski J, Kates SL, Schwarz EM, Gill SR, Zuscik MJ, Mooney RA. 2018. Obesity/type 2 diabetes increases inflammation, periosteal reactive bone formation, and osteolysis during Staphylococcus aureus implant-associated bone infection. J Orthop Res 36:1614–1623. doi: 10.1002/jor.23831. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Horst SA, Hoerr V, Beineke A, Kreis C, Tuchscherr L, Kalinka J, Lehne S, Schleicher I, Kohler G, Fuchs T, Raschke MJ, Rohde M, Peters G, Faber C, Loffler B, Medina E. 2012. A novel mouse model of Staphylococcus aureus chronic osteomyelitis that closely mimics the human infection: an integrated view of disease pathogenesis. Am J Pathol 181:1206–1214. doi: 10.1016/j.ajpath.2012.07.005. [DOI] [PubMed] [Google Scholar]
14.Cheng AG, DeDent AC, Schneewind O, Missiakas D. 2011. A play in four acts: staphylococcus aureus abscess formation. Trends Microbiol 19:225–232. doi: 10.1016/j.tim.2011.01.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Masters EA, Trombetta RP, de Mesy Bentley KL, Boyce BF, Gill AL, Gill SR, Nishitani K, Ishikawa M, Morita Y, Ito H, Bello-Irizarry SN, Ninomiya M, Brodell JD Jr, Lee CC, Hao SP, Oh I, Xie C, Awad HA, Daiss JL, Owen JR, Kates SL, Schwarz EM, Muthukrishnan G. 2019. Evolving concepts in bone infection: redefining “biofilm,” “acute vs. chronic osteomyelitis,” “the immune proteome” and “local antibiotic therapy.” Bone Res 7:20. doi: 10.1038/s41413-019-0061-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Thomer L, Schneewind O, Missiakas D. 2016. Pathogenesis of Staphylococcus aureus bloodstream infections. Annu Rev Pathol 11:343–364. doi: 10.1146/annurev-pathol-012615-044351. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Cheng AG, McAdow M, Kim HK, Bae T, Missiakas DM, Schneewind O. 2010. Contribution of coagulases towards Staphylococcus aureus disease and protective immunity. PLoS Pathog 6:e1001036. doi: 10.1371/journal.ppat.1001036. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Farnsworth CW, Schott EM, Jensen SE, Zukoski J, Benvie AM, Refaai MA, Kates SL, Schwarz EM, Zuscik MJ, Gill SR, Mooney RA. 2017. Adaptive upregulation of clumping factor A (ClfA) by Staphylococcus aureus in the obese, type 2 diabetic host mediates increased virulence. Infect Immun 85:e01005-16. doi: 10.1128/IAI.01005-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Loffler B, Hussain M, Grundmeier M, Bruck M, Holzinger D, Varga G, Roth J, Kahl BC, Proctor RA, Peters G. 2010. Staphylococcus aureus Panton-valentine leukocidin is a very potent cytotoxic factor for human neutrophils. PLoS Pathog 6:e1000715. doi: 10.1371/journal.ppat.1000715. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Okada K, Ueshima S, Tanaka M, Fukao H, Matsuo O. 2000. Analysis of plasminogen activation by the plasmin-staphylokinase complex in plasma of alpha2-antiplasmin-deficient mice. Blood Coagul Fibrinolysis 11:645–655. doi: 10.1097/00001721-200010000-00009. [DOI] [PubMed] [Google Scholar]
21.Vanassche T, Verhaegen J, Peetermans WE, VAN Ryn J, Cheng A, Schneewind O, Hoylaerts MF, Verhamme P. 2011. Inhibition of staphylothrombin by dabigatran reduces Staphylococcus aureus virulence. J Thromb Haemost 9:2436–2446. doi: 10.1111/j.1538-7836.2011.04529.x. [DOI] [PubMed] [Google Scholar]
22.Giai C, Gonzalez CD, Sabbione F, Garofalo A, Ojeda D, Sordelli DO, Trevani AS, Gomez MI. 2016. Staphylococcus aureus induces shedding of IL 1RII in monocytes and neutrophils. J Innate Immun 8:284–298. doi: 10.1159/000443663. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Subbiahdoss G, Fernandez IC, Domingues JF, Kuijer R, van der Mei HC, Busscher HJ. 2011. In vitro interactions between bacteria, osteoblast-like cells and macrophages in the pathogenesis of biomaterial-associated infections. PLoS One 6:e24827. doi: 10.1371/journal.pone.0024827. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Trouillet-Assant S, Gallet M, Nauroy P, Rasigade JP, Flammier S, Parroche P, Marvel J, Ferry T, Vandenesch F, Jurdic P, Laurent F. 2015. Dual impact of live Staphylococcus aureus on the osteoclast lineage, leading to increased bone resorption. J Infect Dis 211:571–581. doi: 10.1093/infdis/jiu386. [DOI] [PubMed] [Google Scholar]
25.Barrila J, Radtke AL, Crabbe A, Sarker SF, Herbst-Kralovetz MM, Ott CM, Nickerson CA. 2010. Organotypic 3D cell culture models: using the rotating wall vessel to study host-pathogen interactions. Nat Rev Microbiol 8:791–801. doi: 10.1038/nrmicro2423. [DOI] [PubMed] [Google Scholar]
26.Bartfeld S, Clevers H. 2015. Organoids as model for infectious diseases: culture of human and murine stomach organoids and microinjection of Helicobacter Pylori. J Vis Exp 105:53359. doi: 10.3791/53359. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Haisma EM, Rietveld MH, de Breij A, van Dissel JT, El Ghalbzouri A, Nibbering PH. 2013. Inflammatory and antimicrobial responses to methicillin-resistant Staphylococcus aureus in an in vitro wound infection model. PLoS One 8:e82800. doi: 10.1371/journal.pone.0082800. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Nickerson CA, Goodwin TJ, Terlonge J, Ott CM, Buchanan KL, Uicker WC, Emami K, LeBlanc CL, Ramamurthy R, Clarke MS, Vanderburg CR, Hammond T, Pierson DL. 2001. Three-dimensional tissue assemblies: novel models for the study of Salmonella enterica serovar Typhimurium pathogenesis. Infect Immun 69:7106–7120. doi: 10.1128/IAI.69.11.7106-7120.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Guggenberger C, Wolz C, Morrissey JA, Heesemann J. 2012. Two distinct coagulase-dependent barriers protect Staphylococcus aureus from neutrophils in a three dimensional in vitro infection model. PLoS Pathog 8:e1002434. doi: 10.1371/journal.ppat.1002434. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Hudson MC, Ramp WK, Frankenburg KP. 1999. Staphylococcus aureus adhesion to bone matrix and bone-associated biomaterials. FEMS Microbiol Lett 173:279–284. doi: 10.1111/j.1574-6968.1999.tb13514.x. [DOI] [PubMed] [Google Scholar]
31.Garzoni C, Kelley WL. 2009. Staphylococcus aureus: new evidence for intracellular persistence. Trends Microbiol 17:59–65. doi: 10.1016/j.tim.2008.11.005. [DOI] [PubMed] [Google Scholar]
32.Crosby HA, Kwiecinski J, Horswill AR. 2016. Staphylococcus aureus Aggregation and coagulation mechanisms, and their function in host-pathogen interactions. Adv Appl Microbiol 96:1–41. doi: 10.1016/bs.aambs.2016.07.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Bjerketorp J, Jacobsson K, Frykberg L. 2004. The von Willebrand factor-binding protein (vWbp) of Staphylococcus aureus is a coagulase. FEMS Microbiol Lett 234:309–314. doi: 10.1016/j.femsle.2004.03.040. [DOI] [PubMed] [Google Scholar]
34.Friedrich R, Panizzi P, Fuentes-Prior P, Richter K, Verhamme I, Anderson PJ, Kawabata S, Huber R, Bode W, Bock PE. 2003. Staphylocoagulase is a prototype for the mechanism of cofactor-induced zymogen activation. Nature 425:535–539. doi: 10.1038/nature01962. [DOI] [PubMed] [Google Scholar]
35.Nair D, Memmi G, Hernandez D, Bard J, Beaume M, Gill S, Francois P, Cheung AL. 2011. Whole-genome sequencing of Staphylococcus aureus strain RN4220, a key laboratory strain used in virulence research, identifies mutations that affect not only virulence factors but also the fitness of the strain. J Bacteriol 193:2332–2335. doi: 10.1128/JB.00027-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Recsei P, Kreiswirth B, O'Reilly M, Schlievert P, Gruss A, Novick RP. 1986. Regulation of exoprotein gene expression in Staphylococcus aureus by agar. Mol Gen Genet 202:58–61. doi: 10.1007/BF00330517. [DOI] [PubMed] [Google Scholar]
37.Kwieciński J, Josefsson E, Mitchell J, Higgins J, Magnusson M, Foster T, Jin T, Bokarewa M. 2010. Activation of plasminogen by staphylokinase reduces the severity of Staphylococcus aureus systemic infection. J Infect Dis 202:1041–1049. doi: 10.1086/656140. [DOI] [PubMed] [Google Scholar]
38.Flemming HC, Wingender J, Szewzyk U, Steinberg P, Rice SA, Kjelleberg S. 2016. Biofilms: an emergent form of bacterial life. Nat Rev Microbiol 14:563–575. doi: 10.1038/nrmicro.2016.94. [DOI] [PubMed] [Google Scholar]
39.Cheng AG, Kim HK, Burts ML, Krausz T, Schneewind O, Missiakas DM. 2009. Genetic requirements for Staphylococcus aureus abscess formation and persistence in host tissues. FASEB J 23:3393–3404. doi: 10.1096/fj.09-135467. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Calhoun JH, Manring MM, Shirtliff M. 2009. Osteomyelitis of the long bones. Semin Plast Surg 23:59–72. doi: 10.1055/s-0029-1214158. [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Dastgheyb S, Parvizi J, Shapiro IM, Hickok NJ, Otto M. 2015. Effect of biofilms on recalcitrance of staphylococcal joint infection to antibiotic treatment. J Infect Dis 211:641–650. doi: 10.1093/infdis/jiu514. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Vanassche T, Peetermans M, Van Aelst LN, Peetermans WE, Verhaegen J, Missiakas DM, Schneewind O, Hoylaerts MF, Verhamme P. 2013. The role of staphylothrombin-mediated fibrin deposition in catheter-related Staphylococcus aureus infections. J Infect Dis 208:92–100. doi: 10.1093/infdis/jit130. [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Mottola C, Matias CS, Mendes JJ, Melo-Cristino J, Tavares L, Cavaco-Silva P, Oliveira M. 2016. Susceptibility patterns of Staphylococcus aureus biofilms in diabetic foot infections. BMC Microbiol 16:119. doi: 10.1186/s12866-016-0737-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Kwiecinski J, Peetermans M, Liesenborghs L, Na M, Björnsdottir H, Zhu X, Jacobsson G, Johansson BR, Geoghegan JA, Foster TJ, Josefsson E, Bylund J, Verhamme P, Jin T. 2016. Staphylokinase control of Staphylococcus aureus biofilm formation and detachment through host plasminogen activation. J Infect Dis 213:139–148. doi: 10.1093/infdis/jiv360. [DOI] [PubMed] [Google Scholar]
45.Konigs AM, Flemming HC, Wingender J. 2015. Nanosilver induces a non-culturable but metabolically active state in Pseudomonas aeruginosa. Front Microbiol 6:395. doi: 10.3389/fmicb.2015.00395. [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Thuptimdang P, Limpiyakorn T, McEvoy J, Prüß BM, Khan E. 2015. Effect of silver nanoparticles on Pseudomonas putida biofilms at different stages of maturity. J Hazard Mater 290:127–133. doi: 10.1016/j.jhazmat.2015.02.073. [DOI] [PubMed] [Google Scholar]
47.Haaber J, Cohn MT, Frees D, Andersen TJ, Ingmer H. 2012. Planktonic aggregates of Staphylococcus aureus protect against common antibiotics. PLoS One 7:e41075. doi: 10.1371/journal.pone.0041075. [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Niemiec MJ, De Samber B, Garrevoet J, Vergucht E, Vekemans B, De Rycke R, Bjorn E, Sandblad L, Wellenreuther G, Falkenberg G, Cloetens P, Vincze L, Urban CF. 2015. Trace element landscape of resting and activated human neutrophils on the sub-micrometer level. Metallomics 7:996–1010. doi: 10.1039/c4mt00346b. [DOI] [PubMed] [Google Scholar]
49.Rincon E, Rocha-Gregg BL, Collins SR. 2018. A map of gene expression in neutrophil-like cell lines. BMC Genomics 19:573. doi: 10.1186/s12864-018-4957-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Rothfork JM, Dessus-Babus S, Van Wamel WJ, Cheung AL, Gresham HD. 2003. Fibrinogen depletion attenuates Staphyloccocus aureus infection by preventing density-dependent virulence gene up-regulation. J Immunol 171:5389–5395. doi: 10.4049/jimmunol.171.10.5389. [DOI] [PubMed] [Google Scholar]
51.Queck SY, Jameson-Lee M, Villaruz AE, Bach TH, Khan BA, Sturdevant DE, Ricklefs SM, Li M, Otto M. 2008. RNAIII-independent target gene control by the agr quorum-sensing system: insight into the evolution of virulence regulation in Staphylococcus aureus. Mol Cell 32:150–158. doi: 10.1016/j.molcel.2008.08.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Otto M. 2014. Phenol-soluble modulins. Int J Med Microbiol 304:164–169. doi: 10.1016/j.ijmm.2013.11.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Oliveira D, Borges A, Simoes M. 2018. Staphylococcus aureus toxins and their molecular activity in infectious diseases. Toxins 10:252. doi: 10.3390/toxins10060252. [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Papayannopoulos V. 2014. Infection: microbial nucleases turn immune cells against each other. Curr Biol 24:R123–R125. doi: 10.1016/j.cub.2013.12.027. [DOI] [PubMed] [Google Scholar]
55.Moriarty TF, Campoccia D, Nees SK, Boure LP, Richards RG. 2010. In vivo evaluation of the effect of intramedullary nail microtopography on the development of local infection in rabbits. Int J Artif Organs 33:667–675. doi: 10.1177/039139881003300913. [DOI] [PubMed] [Google Scholar]
56.Heilmann C, Gerke C, Perdreau-Remington F, Götz F. 1996. Characterization of Tn917 insertion mutants of Staphylococcus epidermidis affected in biofilm formation. Infect Immun 64:277–282. doi: 10.1128/IAI.64.1.277-282.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Pivot-Pajot C, Chouinard FC, El Azreq MA, Harbour D, Bourgoin SG. 2010. Characterisation of degranulation and phagocytic capacity of a human neutrophilic cellular model, PLB-985 cells. Immunobiology 215:38–52. doi: 10.1016/j.imbio.2009.01.007. [DOI] [PubMed] [Google Scholar]
58.Rochford ETJ, Sabate Bresco M, Zeiter S, Kluge K, Poulsson A, Ziegler M, Richards RG, O'Mahony L, Moriarty TF. 2016. Monitoring immune responses in a mouse model of fracture fixation with and without Staphylococcus aureus osteomyelitis. Bone 83:82–92. doi: 10.1016/j.bone.2015.10.014. [DOI] [PubMed] [Google Scholar]
59.Lendrum AC, Fraser DS, Slidders W, Henderson R. 1962. Studies on the character and staining of fibrin. J Clin Pathol 15:401–413. doi: 10.1136/jcp.15.5.401. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental file 1

IAI.00293-20-s0001.pdf^{(868.3KB, pdf)}

[B1] 1.den Heijer CD, van Bijnen EM, Paget WJ, Pringle M, Goossens H, Bruggeman CA, Schellevis FG, Stobberingh EE, APRES Study Team. 2013. Prevalence and resistance of commensal Staphylococcus aureus, including meticillin-resistant S aureus, in nine European countries: a cross-sectional study. Lancet Infect Dis 13:409–415. doi: 10.1016/S1473-3099(13)70036-7. [DOI] [PubMed] [Google Scholar]

[B2] 2.Kluytmans J, van Belkum A, Verbrugh H. 1997. Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clin Microbiol Rev 10:505–520. doi: 10.1128/CMR.10.3.505-520.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B3] 3.Morgenstern M, Erichsen C, Hackl S, Mily J, Militz M, Friederichs J, Hungerer S, Buhren V, Moriarty TF, Post V, Richards RG, Kates SL. 2016. Antibiotic resistance of commensal Staphylococcus aureus and coagulase-negative staphylococci in an international cohort of surgeons: a prospective point-prevalence study. PLoS One 11:e0148437. doi: 10.1371/journal.pone.0148437. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4.Tong SY, Davis JS, Eichenberger E, Holland TL, Fowler VG Jr. 2015. Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev 28:603–661. doi: 10.1128/CMR.00134-14. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B5] 5.Lowy FD. 1998. Staphylococcus aureus infections. N Engl J Med 339:520–532. doi: 10.1056/NEJM199808203390806. [DOI] [PubMed] [Google Scholar]

[B6] 6.Sreeramoju P, Porbandarwalla NS, Arango J, Latham K, Dent DL, Stewart RM, Patterson JE. 2011. Recurrent skin and soft tissue infections due to methicillin-resistant Staphylococcus aureus requiring operative debridement. Am J Surg 201:216–220. doi: 10.1016/j.amjsurg.2009.12.024. [DOI] [PubMed] [Google Scholar]

[B7] 7.Conterno LO, Turchi MD. 2013. Antibiotics for treating chronic osteomyelitis in adults. Cochrane Database Syst Rev 9:CD004439. doi: 10.1002/14651858.CD004439.pub3:CD004439. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8] 8.Bae IG, Tonthat GT, Stryjewski ME, Rude TH, Reilly LF, Barriere SL, Genter FC, Corey GR, Fowler VG Jr. 2009. Presence of genes encoding the Panton-valentine leukocidin exotoxin is not the primary determinant of outcome in patients with complicated skin and skin structure infections due to methicillin-resistant Staphylococcus aureus: results of a multinational trial. J Clin Microbiol 47:3952–3957. doi: 10.1128/JCM.01643-09. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9.Ochsner PE, Hailemariam S. 2006. Histology of osteosynthesis associated bone infection. Injury 37:S49–S58. doi: 10.1016/j.injury.2006.04.009. [DOI] [PubMed] [Google Scholar]

[B10] 10.Miro JM, Anguera I, Cabell CH, Chen AY, Stafford JA, Corey GR, Olaison L, Eykyn S, Hoen B, Abrutyn E, Raoult D, Bayer A, Fowler VG Jr, International Collaboration on Endocarditis Merged Database Study Group. 2005. Staphylococcus aureus native valve infective endocarditis: report of 566 episodes from the International Collaboration on Endocarditis Merged Database. Clin Infect Dis 41:507–514. doi: 10.1086/431979. [DOI] [PubMed] [Google Scholar]

[B11] 11.Brandt SL, Putnam NE, Cassat JE, Serezani CH. 2018. Innate immunity to Staphylococcus aureus: evolving paradigms in soft tissue and invasive infections. J Immunol 200:3871–3880. doi: 10.4049/jimmunol.1701574. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12.Farnsworth CW, Schott EM, Benvie AM, Zukoski J, Kates SL, Schwarz EM, Gill SR, Zuscik MJ, Mooney RA. 2018. Obesity/type 2 diabetes increases inflammation, periosteal reactive bone formation, and osteolysis during Staphylococcus aureus implant-associated bone infection. J Orthop Res 36:1614–1623. doi: 10.1002/jor.23831. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13.Horst SA, Hoerr V, Beineke A, Kreis C, Tuchscherr L, Kalinka J, Lehne S, Schleicher I, Kohler G, Fuchs T, Raschke MJ, Rohde M, Peters G, Faber C, Loffler B, Medina E. 2012. A novel mouse model of Staphylococcus aureus chronic osteomyelitis that closely mimics the human infection: an integrated view of disease pathogenesis. Am J Pathol 181:1206–1214. doi: 10.1016/j.ajpath.2012.07.005. [DOI] [PubMed] [Google Scholar]

[B14] 14.Cheng AG, DeDent AC, Schneewind O, Missiakas D. 2011. A play in four acts: staphylococcus aureus abscess formation. Trends Microbiol 19:225–232. doi: 10.1016/j.tim.2011.01.007. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15.Masters EA, Trombetta RP, de Mesy Bentley KL, Boyce BF, Gill AL, Gill SR, Nishitani K, Ishikawa M, Morita Y, Ito H, Bello-Irizarry SN, Ninomiya M, Brodell JD Jr, Lee CC, Hao SP, Oh I, Xie C, Awad HA, Daiss JL, Owen JR, Kates SL, Schwarz EM, Muthukrishnan G. 2019. Evolving concepts in bone infection: redefining “biofilm,” “acute vs. chronic osteomyelitis,” “the immune proteome” and “local antibiotic therapy.” Bone Res 7:20. doi: 10.1038/s41413-019-0061-z. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16.Thomer L, Schneewind O, Missiakas D. 2016. Pathogenesis of Staphylococcus aureus bloodstream infections. Annu Rev Pathol 11:343–364. doi: 10.1146/annurev-pathol-012615-044351. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B17] 17.Cheng AG, McAdow M, Kim HK, Bae T, Missiakas DM, Schneewind O. 2010. Contribution of coagulases towards Staphylococcus aureus disease and protective immunity. PLoS Pathog 6:e1001036. doi: 10.1371/journal.ppat.1001036. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B18] 18.Farnsworth CW, Schott EM, Jensen SE, Zukoski J, Benvie AM, Refaai MA, Kates SL, Schwarz EM, Zuscik MJ, Gill SR, Mooney RA. 2017. Adaptive upregulation of clumping factor A (ClfA) by Staphylococcus aureus in the obese, type 2 diabetic host mediates increased virulence. Infect Immun 85:e01005-16. doi: 10.1128/IAI.01005-16. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B19] 19.Loffler B, Hussain M, Grundmeier M, Bruck M, Holzinger D, Varga G, Roth J, Kahl BC, Proctor RA, Peters G. 2010. Staphylococcus aureus Panton-valentine leukocidin is a very potent cytotoxic factor for human neutrophils. PLoS Pathog 6:e1000715. doi: 10.1371/journal.ppat.1000715. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B20] 20.Okada K, Ueshima S, Tanaka M, Fukao H, Matsuo O. 2000. Analysis of plasminogen activation by the plasmin-staphylokinase complex in plasma of alpha2-antiplasmin-deficient mice. Blood Coagul Fibrinolysis 11:645–655. doi: 10.1097/00001721-200010000-00009. [DOI] [PubMed] [Google Scholar]

[B21] 21.Vanassche T, Verhaegen J, Peetermans WE, VAN Ryn J, Cheng A, Schneewind O, Hoylaerts MF, Verhamme P. 2011. Inhibition of staphylothrombin by dabigatran reduces Staphylococcus aureus virulence. J Thromb Haemost 9:2436–2446. doi: 10.1111/j.1538-7836.2011.04529.x. [DOI] [PubMed] [Google Scholar]

[B22] 22.Giai C, Gonzalez CD, Sabbione F, Garofalo A, Ojeda D, Sordelli DO, Trevani AS, Gomez MI. 2016. Staphylococcus aureus induces shedding of IL 1RII in monocytes and neutrophils. J Innate Immun 8:284–298. doi: 10.1159/000443663. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B23] 23.Subbiahdoss G, Fernandez IC, Domingues JF, Kuijer R, van der Mei HC, Busscher HJ. 2011. In vitro interactions between bacteria, osteoblast-like cells and macrophages in the pathogenesis of biomaterial-associated infections. PLoS One 6:e24827. doi: 10.1371/journal.pone.0024827. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24.Trouillet-Assant S, Gallet M, Nauroy P, Rasigade JP, Flammier S, Parroche P, Marvel J, Ferry T, Vandenesch F, Jurdic P, Laurent F. 2015. Dual impact of live Staphylococcus aureus on the osteoclast lineage, leading to increased bone resorption. J Infect Dis 211:571–581. doi: 10.1093/infdis/jiu386. [DOI] [PubMed] [Google Scholar]

[B25] 25.Barrila J, Radtke AL, Crabbe A, Sarker SF, Herbst-Kralovetz MM, Ott CM, Nickerson CA. 2010. Organotypic 3D cell culture models: using the rotating wall vessel to study host-pathogen interactions. Nat Rev Microbiol 8:791–801. doi: 10.1038/nrmicro2423. [DOI] [PubMed] [Google Scholar]

[B26] 26.Bartfeld S, Clevers H. 2015. Organoids as model for infectious diseases: culture of human and murine stomach organoids and microinjection of Helicobacter Pylori. J Vis Exp 105:53359. doi: 10.3791/53359. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27.Haisma EM, Rietveld MH, de Breij A, van Dissel JT, El Ghalbzouri A, Nibbering PH. 2013. Inflammatory and antimicrobial responses to methicillin-resistant Staphylococcus aureus in an in vitro wound infection model. PLoS One 8:e82800. doi: 10.1371/journal.pone.0082800. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B28] 28.Nickerson CA, Goodwin TJ, Terlonge J, Ott CM, Buchanan KL, Uicker WC, Emami K, LeBlanc CL, Ramamurthy R, Clarke MS, Vanderburg CR, Hammond T, Pierson DL. 2001. Three-dimensional tissue assemblies: novel models for the study of Salmonella enterica serovar Typhimurium pathogenesis. Infect Immun 69:7106–7120. doi: 10.1128/IAI.69.11.7106-7120.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B29] 29.Guggenberger C, Wolz C, Morrissey JA, Heesemann J. 2012. Two distinct coagulase-dependent barriers protect Staphylococcus aureus from neutrophils in a three dimensional in vitro infection model. PLoS Pathog 8:e1002434. doi: 10.1371/journal.ppat.1002434. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B30] 30.Hudson MC, Ramp WK, Frankenburg KP. 1999. Staphylococcus aureus adhesion to bone matrix and bone-associated biomaterials. FEMS Microbiol Lett 173:279–284. doi: 10.1111/j.1574-6968.1999.tb13514.x. [DOI] [PubMed] [Google Scholar]

[B31] 31.Garzoni C, Kelley WL. 2009. Staphylococcus aureus: new evidence for intracellular persistence. Trends Microbiol 17:59–65. doi: 10.1016/j.tim.2008.11.005. [DOI] [PubMed] [Google Scholar]

[B32] 32.Crosby HA, Kwiecinski J, Horswill AR. 2016. Staphylococcus aureus Aggregation and coagulation mechanisms, and their function in host-pathogen interactions. Adv Appl Microbiol 96:1–41. doi: 10.1016/bs.aambs.2016.07.018. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B33] 33.Bjerketorp J, Jacobsson K, Frykberg L. 2004. The von Willebrand factor-binding protein (vWbp) of Staphylococcus aureus is a coagulase. FEMS Microbiol Lett 234:309–314. doi: 10.1016/j.femsle.2004.03.040. [DOI] [PubMed] [Google Scholar]

[B34] 34.Friedrich R, Panizzi P, Fuentes-Prior P, Richter K, Verhamme I, Anderson PJ, Kawabata S, Huber R, Bode W, Bock PE. 2003. Staphylocoagulase is a prototype for the mechanism of cofactor-induced zymogen activation. Nature 425:535–539. doi: 10.1038/nature01962. [DOI] [PubMed] [Google Scholar]

[B35] 35.Nair D, Memmi G, Hernandez D, Bard J, Beaume M, Gill S, Francois P, Cheung AL. 2011. Whole-genome sequencing of Staphylococcus aureus strain RN4220, a key laboratory strain used in virulence research, identifies mutations that affect not only virulence factors but also the fitness of the strain. J Bacteriol 193:2332–2335. doi: 10.1128/JB.00027-11. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B36] 36.Recsei P, Kreiswirth B, O'Reilly M, Schlievert P, Gruss A, Novick RP. 1986. Regulation of exoprotein gene expression in Staphylococcus aureus by agar. Mol Gen Genet 202:58–61. doi: 10.1007/BF00330517. [DOI] [PubMed] [Google Scholar]

[B37] 37.Kwieciński J, Josefsson E, Mitchell J, Higgins J, Magnusson M, Foster T, Jin T, Bokarewa M. 2010. Activation of plasminogen by staphylokinase reduces the severity of Staphylococcus aureus systemic infection. J Infect Dis 202:1041–1049. doi: 10.1086/656140. [DOI] [PubMed] [Google Scholar]

[B38] 38.Flemming HC, Wingender J, Szewzyk U, Steinberg P, Rice SA, Kjelleberg S. 2016. Biofilms: an emergent form of bacterial life. Nat Rev Microbiol 14:563–575. doi: 10.1038/nrmicro.2016.94. [DOI] [PubMed] [Google Scholar]

[B39] 39.Cheng AG, Kim HK, Burts ML, Krausz T, Schneewind O, Missiakas DM. 2009. Genetic requirements for Staphylococcus aureus abscess formation and persistence in host tissues. FASEB J 23:3393–3404. doi: 10.1096/fj.09-135467. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B40] 40.Calhoun JH, Manring MM, Shirtliff M. 2009. Osteomyelitis of the long bones. Semin Plast Surg 23:59–72. doi: 10.1055/s-0029-1214158. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B41] 41.Dastgheyb S, Parvizi J, Shapiro IM, Hickok NJ, Otto M. 2015. Effect of biofilms on recalcitrance of staphylococcal joint infection to antibiotic treatment. J Infect Dis 211:641–650. doi: 10.1093/infdis/jiu514. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B42] 42.Vanassche T, Peetermans M, Van Aelst LN, Peetermans WE, Verhaegen J, Missiakas DM, Schneewind O, Hoylaerts MF, Verhamme P. 2013. The role of staphylothrombin-mediated fibrin deposition in catheter-related Staphylococcus aureus infections. J Infect Dis 208:92–100. doi: 10.1093/infdis/jit130. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B43] 43.Mottola C, Matias CS, Mendes JJ, Melo-Cristino J, Tavares L, Cavaco-Silva P, Oliveira M. 2016. Susceptibility patterns of Staphylococcus aureus biofilms in diabetic foot infections. BMC Microbiol 16:119. doi: 10.1186/s12866-016-0737-0. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B44] 44.Kwiecinski J, Peetermans M, Liesenborghs L, Na M, Björnsdottir H, Zhu X, Jacobsson G, Johansson BR, Geoghegan JA, Foster TJ, Josefsson E, Bylund J, Verhamme P, Jin T. 2016. Staphylokinase control of Staphylococcus aureus biofilm formation and detachment through host plasminogen activation. J Infect Dis 213:139–148. doi: 10.1093/infdis/jiv360. [DOI] [PubMed] [Google Scholar]

[B45] 45.Konigs AM, Flemming HC, Wingender J. 2015. Nanosilver induces a non-culturable but metabolically active state in Pseudomonas aeruginosa. Front Microbiol 6:395. doi: 10.3389/fmicb.2015.00395. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B46] 46.Thuptimdang P, Limpiyakorn T, McEvoy J, Prüß BM, Khan E. 2015. Effect of silver nanoparticles on Pseudomonas putida biofilms at different stages of maturity. J Hazard Mater 290:127–133. doi: 10.1016/j.jhazmat.2015.02.073. [DOI] [PubMed] [Google Scholar]

[B47] 47.Haaber J, Cohn MT, Frees D, Andersen TJ, Ingmer H. 2012. Planktonic aggregates of Staphylococcus aureus protect against common antibiotics. PLoS One 7:e41075. doi: 10.1371/journal.pone.0041075. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B48] 48.Niemiec MJ, De Samber B, Garrevoet J, Vergucht E, Vekemans B, De Rycke R, Bjorn E, Sandblad L, Wellenreuther G, Falkenberg G, Cloetens P, Vincze L, Urban CF. 2015. Trace element landscape of resting and activated human neutrophils on the sub-micrometer level. Metallomics 7:996–1010. doi: 10.1039/c4mt00346b. [DOI] [PubMed] [Google Scholar]

[B49] 49.Rincon E, Rocha-Gregg BL, Collins SR. 2018. A map of gene expression in neutrophil-like cell lines. BMC Genomics 19:573. doi: 10.1186/s12864-018-4957-6. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B50] 50.Rothfork JM, Dessus-Babus S, Van Wamel WJ, Cheung AL, Gresham HD. 2003. Fibrinogen depletion attenuates Staphyloccocus aureus infection by preventing density-dependent virulence gene up-regulation. J Immunol 171:5389–5395. doi: 10.4049/jimmunol.171.10.5389. [DOI] [PubMed] [Google Scholar]

[B51] 51.Queck SY, Jameson-Lee M, Villaruz AE, Bach TH, Khan BA, Sturdevant DE, Ricklefs SM, Li M, Otto M. 2008. RNAIII-independent target gene control by the agr quorum-sensing system: insight into the evolution of virulence regulation in Staphylococcus aureus. Mol Cell 32:150–158. doi: 10.1016/j.molcel.2008.08.005. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B52] 52.Otto M. 2014. Phenol-soluble modulins. Int J Med Microbiol 304:164–169. doi: 10.1016/j.ijmm.2013.11.019. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B53] 53.Oliveira D, Borges A, Simoes M. 2018. Staphylococcus aureus toxins and their molecular activity in infectious diseases. Toxins 10:252. doi: 10.3390/toxins10060252. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B54] 54.Papayannopoulos V. 2014. Infection: microbial nucleases turn immune cells against each other. Curr Biol 24:R123–R125. doi: 10.1016/j.cub.2013.12.027. [DOI] [PubMed] [Google Scholar]

[B55] 55.Moriarty TF, Campoccia D, Nees SK, Boure LP, Richards RG. 2010. In vivo evaluation of the effect of intramedullary nail microtopography on the development of local infection in rabbits. Int J Artif Organs 33:667–675. doi: 10.1177/039139881003300913. [DOI] [PubMed] [Google Scholar]

[B56] 56.Heilmann C, Gerke C, Perdreau-Remington F, Götz F. 1996. Characterization of Tn917 insertion mutants of Staphylococcus epidermidis affected in biofilm formation. Infect Immun 64:277–282. doi: 10.1128/IAI.64.1.277-282.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B57] 57.Pivot-Pajot C, Chouinard FC, El Azreq MA, Harbour D, Bourgoin SG. 2010. Characterisation of degranulation and phagocytic capacity of a human neutrophilic cellular model, PLB-985 cells. Immunobiology 215:38–52. doi: 10.1016/j.imbio.2009.01.007. [DOI] [PubMed] [Google Scholar]

[B58] 58.Rochford ETJ, Sabate Bresco M, Zeiter S, Kluge K, Poulsson A, Ziegler M, Richards RG, O'Mahony L, Moriarty TF. 2016. Monitoring immune responses in a mouse model of fracture fixation with and without Staphylococcus aureus osteomyelitis. Bone 83:82–92. doi: 10.1016/j.bone.2015.10.014. [DOI] [PubMed] [Google Scholar]

[B59] 59.Lendrum AC, Fraser DS, Slidders W, Henderson R. 1962. Studies on the character and staining of fibrin. J Clin Pathol 15:401–413. doi: 10.1136/jcp.15.5.401. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Three-Dimensional In Vitro Staphylococcus aureus Abscess Communities Display Antibiotic Tolerance and Protection from Neutrophil Clearance

Marloes I Hofstee

Martijn Riool

Igors Terjajevs

Keith Thompson

Martin J Stoddart

R Geoff Richards

Sebastian A J Zaat

T Fintan Moriarty

Roles

ABSTRACT

INTRODUCTION

RESULTS

Morphological characteristics of the 3D in vitro SAC model.

FIG 1.

Growth of in vitro SACs.

FIG 2.

SAC formation by multiple S. aureus strains and clinical isolates.

FIG 3.

Antibiotic tolerance of in vitro SACs.

FIG 4.

Interactions of dPLB cells with in vitro SACs.

FIG 5.

Interactions of primary human neutrophils with in vitro SACs.

FIG 6.

Morphological comparison of in vivo and in vitro SACs.

FIG 7.

DISCUSSION

Conclusions.

MATERIALS AND METHODS

Bacteria.

General SAC methodology.

Host cells.

Antibiotic tolerance of in vitro SACs.

Interactions of dPLB cells or primary human neutrophils with in vitro SACs.

In vivo samples.

Stainings.

Microscopy.

Statistical analysis.

Supplementary Material

ACKNOWLEDGMENTS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases