Survival of Staphylococcus epidermidis in Fibroblasts andOsteoblasts

Kimberly Perez,a Robin Patelb,c

aDepartment of Immunology, Mayo Clinic, Rochester, Minnesota, USAbDivision of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester,Minnesota, USA

cDivision of Infectious Diseases, Department of Medicine, Mayo Clinic, Rochester, Minnesota, USA

ABSTRACT Staphylococcus epidermidis is a leading cause of infections associatedwith indwelling medical devices, including prosthetic joint infection. While biofilmformation is assumed to be the main mechanism underlying the chronic infectionsS. epidermidis causes, we hypothesized that S. epidermidis also evades immune kill-ing, contributing to its pathogenesis. Here, we show that prosthetic joint-associatedS. epidermidis isolates can persist intracellularly within human fibroblasts and insidehuman and mouse osteoblasts. We also show that the intracellularly persisting bac-teria reside primarily within acidic phagolysosomes and that over the course of in-fection, small-colony variants are selected for. Moreover, upon eukaryotic cell death,these bacteria, which can outlive their host, can escape into the extracellular envi-ronment, providing them an opportunity to form biofilms on implant surfaces at de-layed time points in implant-associated infection. In summary, the acidic phagolyso-somes of fibroblasts and osteoblasts serve as reservoirs for chronic or delayed S.epidermidis infection.

KEYWORDS fibroblast, intracellular infection, osteoblast, Staphylococcus epidermidis

Staphylococcus epidermidis is a generally nonvirulent Gram-positive bacteriumwhich is a member of the normal human skin microbiota. Similar to many gut

bacteria, this commensal organism typically serves a beneficial role, educating theimmune system and allowing immune cells to keep the potentially harmful micro-biota in check while not mounting an inflammatory response (1–5). As an example,S. epidermidis produces phenol-soluble modulin (PSM) peptides, present on thesurface of the human skin (1), which have antimicrobial activity against somepathogenic bacteria, including Staphylococcus aureus and Streptococcus pyogenes(6). In addition, S. epidermidis stimulates keratinocytes to produce antimicrobialpeptides which inhibit the growth of S. aureus and S. pyogenes (2). Despite itsnormally constructive role, with the advent of implantation of medical devices, S.epidermidis has emerged as an important foreign body-associated pathogen and isnow, for example, one of the leading causes of prosthetic joint infection (PJI) (7–11).Although S. epidermidis infection does not frequently lead to death, the chronicinfection it causes contributes to a high economic burden and morbidity (8–12). In2015, an estimated 1.3 million hip and knee replacement procedures were per-formed in the United States, with numbers estimated to double by 2020 (13).Infection rates have held steady at 2 to 2.4% and lead to an annual cost projectedto reach 1.62 billion dollars by 2020 (13). Together, S. aureus and S. epidermidis arethe causative agents for more than half of all PJI cases. Given the steadily increasingnumbers of joint arthroplasty surgeries being performed, and accordingly thenumber of associated infections, it is important to define their pathogenesis.

Received 30 March 2018 Returned formodification 31 May 2018 Accepted 20 July2018

Accepted manuscript posted online 30 July2018

Citation Perez K, Patel R. 2018. Survival ofStaphylococcus epidermidis in fibroblasts andosteoblasts. Infect Immun 86:e00237-18.https://doi.org/10.1128/IAI.00237-18.

Editor Nancy E. Freitag, University of Illinois atChicago

Copyright © 2018 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Robin Patel,patel.robin@mayo.edu.

BACTERIAL INFECTIONS

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Many specific mechanisms of immune evasion have been described for S. aureus,including the production of several factors that inhibit complement activation, com-promise the activity of neutrophils and macrophages, lyse neutrophils, neutralizeantimicrobial peptides (AMPs), and allow for both survival in and escape from phago-somes (12, 14–23). In contrast, there is less known about S. epidermidis pathogenicity.S. epidermidis does not express many of the virulence factors that S. aureus does, andbiofilm formation has been traditionally considered its primary mechanism of patho-genesis when it establishes implant-associated infection (10, 24, 25). In addition toserving as a barrier, biofilm formation has also been shown to decrease deposition ofC3b and IgG, both of which help facilitate phagocytosis and killing by polymorphonu-clear leukocytes (PMNs), on bacterial surfaces (26). Moreover, polysaccharide intercel-lular adhesin (PIA), extracellular matrix-binding protein (Embp), and accumulation-associated protein (Aap), found on the surface of S. epidermidis bacteria, play roles inintercellular adhesion (27–29) and protect biofilms from macrophage phagocytosis (30).

Although existing in a biofilm may enable S. epidermidis to escape the host immunesystem and remain relatively undetected, this is unlikely to be the sole reason that thisorganism is such a prominent cause of implant-associated infections, because manybacteria are capable of biofilm formation. In addition, if biofilm formation were the onlymechanism involved in infection, implant removal might be expected to be universallycurative. In this regard, immune evasion may be operative. Factors found to play rolesin immune evasion by S. epidermidis include its aps and sepA loci. The former senses thepresence of human AMPs, while the latter destroys them (31). Further, the staphylo-coccal quorum sensing system, accessory gene regulator (agr), has been suggested toplay a role in resistance to PMN killing and resistance to the PMN antimicrobial products(26, 32). agr regulates many colonization and virulence factors; one category ofvirulence factors upregulated by agr is PSMs. PSMs are amphipathic peptides producedby most staphylococci that have been demonstrated to play important roles in S. aureusinfection (33) by lysing neutrophils, altering dendritic cell function, and skewing T cellpriming (1, 14, 17, 31, 34, 35). Although S. epidermidis has the capacity to producecytolytic PSMs, it produces such low levels of these peptides that it has little to noability to lyse PMNs (31).

We hypothesized there to be an additional mechanism that S. epidermidis uses tocause chronic infection. Based on the demonstration that S. aureus can invade andpersist in a myriad of cell types (36–47), we hypothesized that S. epidermidis also evadesimmune killing by persisting within nonimmune cells located around implants. Previ-ously it has been reported that a reference strain not associated with human diseaseand two S. epidermidis strains isolated from peritonitis and osteomyelitis cases can beinternalized in MG63 bone cells (48). In addition, we have shown that clinical S.epidermidis PJI isolates can be internalized by and persist within human fibroblasts (49).

The aim of this study was to further investigate intracellular persistence as anothermeans, besides biofilm formation, that S. epidermidis may employ to escape innateimmune killing and cause chronic infection. As several cell types, including fibroblastsand osteoblasts, can be exposed to pathogens in device-related infections, we studiedthe interactions of these cells with S. epidermidis. We evaluated the ability of clinical S.epidermidis PJI isolates to persist intracellularly and to escape host cells using in vitrocell culture infection models, flow cytometry, fluorescence microscopy, and spectrom-etry.

RESULTSS. epidermidis invasion of and persistence in fibroblasts and osteoblasts in

vitro. Planktonic S. epidermidis clinical isolates are readily killed by human neutrophils(see Fig. S1 in the supplemental material), suggesting that some form of evasion of thiskilling is needed to allow for chronic infection. To test the hypothesis that S. epidermidiscan invade and persist within nonprofessional phagocytic cells normally found at thesite of joint arthroplasty, a lysostaphin/daptomycin protection assay was used to assayintracellular infection. Fibroblasts, commonly found at the site of implants due to their

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role in wound healing, and osteoblasts were examined. S. aureus strain 6850, previouslyshown to persist intracellularly, was used as a positive control. Two hours postinfection(p.i.), S. epidermidis strain RP62A and PJI isolates IDRL-8933 and IDRL-8864 were foundto invade all host cell types tested (Fig. 1A). Differences were observed in the numbersof intracellular bacteria taken up by each cell type (Fig. 1A), with mouse osteoblaststaking up more S. aureus 6850, RP62A, and IDRL-8933 bacteria than the other cell typesat 2 h p.i. There were no significant differences in the numbers of bacteria recovered foreach strain at days 0 and 3 p.i. in human fibroblasts (Fig. 1A). Conversely, in primaryhuman osteoblasts, the numbers of CFU recovered were lower for S. aureus 6850 andthe two PJI isolates (IDRL-8933 and IDRL-8864) at day 3 day p.i. than at day 0 (P � 0.002,P � 0.0035, and P � 0.0023, respectively), suggesting microbicidal activity of humanosteoblasts (Fig. 1A). Similarly, in mouse osteoblasts, the numbers of CFU recoveredwere lower for S. aureus 6850 and the two PJI isolates at day 3 day p.i. than at day 0 (P �

0.01, P � 0.0055, and P � 0.0022, respectively) (Fig. 1A).Next, the persistence of each strain in each of the cell types was examined. The two

strains isolated from sepsis-related infections, S. aureus 6850 and S. epidermidis RP62A,were able to survive intracellularly for at least 10 days after infection (Fig. 1B to D).Although the two PJI isolates were also able to persist in human fibroblasts and mouseosteoblasts for the duration of the experiment, by day 5 p.i., no viable bacteria weredetected by quantitative or qualitative culture in human osteoblasts. A similar but

FIG 1 Intracellular persistence of S. epidermidis in nonprofessional phagocytes. S. aureus strain 6850, S.epidermidis strain RP62A, and two S. epidermidis prosthetic joint infection isolates, IDRL-8933 andIDRL-8864, were used to infect MRC5 (human fibroblasts), NHOst (normal human osteoblasts), andMC3T3 (murine osteoblasts) at a multiplicity of infection of 25 for 2 h, after which lysostaphin (25 �g/ml)or daptomycin (100 �g/ml) was added and intracellular bacterial growth was monitored at 0, 3, 5, 7, and10 days postinfection (p.i.). (A) CFU recovered from each host cell type 2 h p.i. and 3 days p.i. *, P � 0.05,**, P � 0.01, ***, P � 0.001. (B to G) CFU recovered from infected human fibroblasts (B), humanosteoblasts (C), and mouse osteoblasts (D) at indicated time points, and percentages of viable humanfibroblasts (E), human osteoblasts (F), and mouse osteoblasts (G) over the course of infection. Data aremean values (�standard deviations of the means) from at least three independent experiments.

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delayed trend was also seen during infection of mouse osteoblasts. To evaluate fordifferences in cytotoxicity among the strains tested, trypan blue exclusion was used toassess host cell death. At 2 h p.i., each strain tested induced less than 20% cell death(Fig. 1E to G), with at least 50% of the population persisting throughout the course ofinfection.

Neither agr nor PSMs are required for invasion or survival during intracellularinfection. To assess whether agr or PSMs are required for invasion and intracellularpersistence, a PSM� deletion mutant and an agr deletion mutant, and their correspond-ing wild-type (wt) strains, 1457 and Tü3298, respectively, were used to infect humanfibroblasts (Fig. S2A). Each of the S. epidermidis strains was able to invade and persistfor at least 7 days p.i., and there were no significant differences in the numbers of viableCFU recovered between the wt and deletion mutant pairs over the course of infection.There were also no differences in associated host cell death (Fig. S2B).

Lack of difference in uptake and intracellular persistence of S. aureus acute-and chronic-infection PJI isolates. Resch et al. have shown that there are differencesin gene expression between S. aureus planktonic and biofilm cells. They demonstratedupregulation of genes encoding toxins in planktonic cells and upregulation of genesassociated with the stress response and cell surface proteins in biofilm cells (50). As ithas been suggested that planktonic S. aureus cells are associated with acute infections,whereas biofilm cells are associated with chronic infections, we studied one S. aureusisolate each associated with chronic and acute PJI to examine for differences ininvasiveness between the two isolates upon infection of mouse osteoblasts. IDRL-9072(chronic PJI associated) and IDRL-11537 (acute PJI associated) invaded and persisted inosteoblasts for at least 7 days p.i., at similar levels as S. aureus 6850, inducing �40% cellhost death over the course of infection (Fig. S2C and D).

Intracellular persistence is accompanied by increased numbers of SCVs. Small-colony variants (SCVs) are slow-growing subpopulations that naturally arise in responseto various environmental pressures (51–53). They normally comprise a minor fraction ofthe source population and are commonly described in chronic S. aureus infections(54–58). Some SCV observations have also been described for S. epidermidis, mostly inassociation with foreign-body-related infections, such as PJI (8, 59–64). Previously, wehave shown that intracellular localization induces S. epidermidis SCV formation inhuman lung fibroblasts (49). To determine if these findings extend to intracellularpersistence in other cell types, we assessed colony phenotypes of viable bacteria afteran overnight incubation on sheep blood agar (SBA) at 37°C, followed by an additionalincubation overnight at room temperature. SCVs were identified on the basis of theirsize (Fig. 2A). At 2 h p.i., an average of 3, 24, and 7% of all viable intracellularly localizedbacteria had a SCV phenotype after infection in human fibroblasts, human osteoblasts,and mouse osteoblasts, respectively (Fig. 2B to D). While the number of viable intra-cellularly persisting bacteria remained fairly constant over the course of infection, thefrequency of SCVs increased in the intracellular environment, reaching an average of24% after 7 days in the human fibroblasts (Fig. 2B). Although the number of viablepersisting bacteria declined over time in the human and mouse osteoblasts, thefrequency of SCVs continued to increase over time, reaching an average of 19 and 25%after 10 days in the human osteoblasts and mouse osteoblasts, respectively (Fig. 2C andD). The drop observed in the average percentage of SCVs in the human osteoblasts wasdue to the death of the two PJI isolates.

Next, to assess whether SCVs have an advantage in intracellular invasiveness, wecompared the numbers of CFU recovered after infection with two normal-colony-phenotype (NCP) isolates and their SCV counterparts recovered from chronic knee andshoulder PJI. There were no statistically significant differences in the numbers ofrecovered bacteria between the NCP and SCV pairs at 2 h p.i. (Fig. S2C), suggesting thatSCVs do not have an increased ability to invade cells in comparison to NCP isolates, aspreviously shown for one S. aureus NCP and SCV pair (65).

Intracellularly persisting S. epidermidis bacteria colocalize with host cellphagolysosomes. Previously, we have shown that S. epidermidis RP62A is located

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within the phagolysosomes of human fibroblasts (49). Here, we aimed to confirm thatthese findings are not limited to a single strain and to evaluate whether intracellularlypersisting S. epidermidis bacteria are also located in phagolysosomes within humanosteoblasts. CFSE (carboxyfluorescein succinimidyl ester)-labeled RP62A colocalizedwith LAMP-2� vesicles in both fixed human fibroblasts and osteoblasts (Fig. 3A and B).Similarly, S. epidermidis 1457-green fluorescent protein (GFP) colocalized with phagoly-sosomes in live cells (Fig. 3C and D). Colocalizations were supported by analysis ofManders’ coefficient. Manders’ coefficient M1 denotes the fraction of phagolysosomesthat colocalize with bacteria, whereas Manders’ coefficient M2 denotes the fraction ofbacteria that colocalize with phagolysosomes. Because there is an excess number ofphagolysosomes compared to bacteria, Manders’ coefficient M1 may be skewed, falselyindicating low colocalization. Thus, we ascribed increased significance to Manders’coefficient M2 in terms of quantifying colocalization. The average Manders’ coefficientM2 values for the two S. epidermidis isolates with the two different phagolysosomalstains were all �0.9, suggesting that intracellularly persisting bacteria are locatedwithin phagolysosomes in both human fibroblasts and osteoblasts (Fig. 3E). Thesefindings were similar for live cells infected with the USA300 strain LAC expressing GFP(LAC-GFP) 3 h p.i. (Fig. S3).

S. epidermidis RP62A and 1457 do not escape the phagolysosome of infectedcells. It has previously been shown that some S. aureus strains are capable of escapingfrom the phagosomal compartment to avoid phagolysosomal killing (15, 40, 44, 66–68).To address the question as to whether escape from the phagolysosome is a mechanismS. epidermidis uses to persist in nonprofessional phagocytes, we used a flow cytometry-based pH monitoring assay described by Lâm et al. (40). At 2 h p.i., all examined strainswere located within an acidic environment (Fig. 4A to C). Differences in arbitraryfluorescent units (ΔAFU) at 6 h p.i. indicated translocation of the two S. aureus strainstested (Fig. 4A to C), previously shown to escape the phagosome in HeLa, 293, andTHP-1 cells (40, 66). The escape of S. aureus strains 6850 and LAC-GFP was indicated bysignificant changes in ΔAFU between the 2- and 6-h time points (for 6850, P � 0.007,P � 0.006, P � 0.007; for LAC-GFP, P � 0.02, P � 0.005, P � 0.0007 [for MRC5 {humanfibroblasts}, NHOst {normal human osteoblasts}, and MC3T3 {murine osteoblasts},respectively]). However, both RP62A and 1457-GFP remained in the endosomal com-

FIG 2 Small-colony variant (SCV) formation increases over time during intracellular persistence. Colonyphenotypes of viable bacteria were assessed after an overnight incubation on sheep blood agar at 37°C,followed by an additional incubation overnight at room temperature. SCVs were identified on the basisof their size. (A) Representative images of colony phenotype of each strain; SCVs are denoted byarrowheads. (B to D) Percentages of viable intracellularly persisting bacteria with SCV phenotype inhuman fibroblasts (B), human osteoblasts (C), and mouse osteoblasts (D) over the course of infection.Data are mean values (�standard deviations of the means) from two independent experiments per-formed in triplicate.

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partment (Fig. 4A to C). At 6 h p.i., the ΔAFU values were lower in the escaping strainsthan in the S. epidermidis strains RP62A (P � 0.001, P � 0.01, P � 0.01) and 1457 (P �

0.0001, P � 0.01, P � 0.01).Osteoblasts and fibroblasts have differential responses to intracellular infec-

tion. To further confirm that S. epidermidis does not escape the phagolysosome, assuggested by the flow cytometry-based assay, we performed live time-lapse micros-copy to monitor bacteria in real time in the intracellular milieu. From 4 to 7.5 h p.i., S.epidermidis 1457 remained in the lysosomal compartment of human fibroblasts (VideoS1). Z-stack imaging and the Manders’ coefficients supported this finding (Fig. 5). Inhuman osteoblasts, time-lapse microscopy revealed these cells to be sensitive tobacterial infection, with cell death observed at less than 5 h p.i. (Video S2). Both nuclearfading and loss of lysosomal staining indicate apoptosis of the host cell at 5.5 h p.i. inthe case of osteoblasts (Fig. 6A and B). The average colocalization coefficients of S.epidermidis 1457-GFP and the phagolysosome from 3.5 to 4 h p.i. provide support that

FIG 3 Intracellularly persisting S. epidermidis bacteria are located within phagolysosomes in both humanfibroblasts and human osteoblasts. Host cells were infected with CFSE-labeled S. epidermidis RP62A or S.epidermidis 1457 expressing superfolder green fluorescent protein (sGFP) (multiplicity of infection of 50).Fixed cell phagolysosomes were visualized with LAMP-2 antibody (Alexa Fluor 594; red), and nuclei werestained with DAPI (4=,6-diamidino-2-phenylindole; blue). Live cell phagolysosomes were visualized usinga CytoPainter lysosomal staining kit (ab112137; red), and Hoechst 33342 was used to stain nuclei. Imageswere acquired using confocal microscopy; arrowheads indicate examples of bacterial colocalization withphagolysosomes. (A and B) Representative images of fixed MRC5 (human fibroblast) (A) and NHOst(human osteoblast) (B) cells that were infected with S. epidermidis RP62A. (C and D) Representativeimages of live human fibroblasts (C) and human osteoblasts (D) that were infected with S. epidermidis1457-GFP. (E) Quantification of the Manders’ colocalization coefficient M1 (fraction of phagolysosomalmarker in colocalization with bacteria) or M2 (fraction of bacteria in colocalization with phagolysosomalmarkers). Data are expressed as mean values (�standard deviations of the mean).

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the bacteria are located within the phagosome (Fig. 6C). There are no coefficients for5 h p.i. due to the lack of colocalization found, but the continual GFP expressionindicates that S. epidermidis 1457-GFP outlived its captor. Together, the time-lapseexperiments provide evidence that S. epidermidis cannot actively escape the phagoly-sosome. However, we show that this organism can remain viable and be released upondeath of the host cell.

To compare the nature of infection between S. aureus and S. epidermidis, S. aureusLAC-GFP was also visualized during intracellular persistence in human fibroblasts andosteoblasts. LAC-GFP was found to escape lysosomal compartments in fibroblasts priorto 6 h p.i. and to replicate in the cytosol in a relatively short period of time (Fig. S4 andVideo S3). This corroborates the previous findings of Grosz et al. that S. aureus replicateswithin the host cell cytoplasm (66). The high standard deviation (SD) seen in the M2coefficient likely reflects the escape of some LAC-GFP bacteria and the replication ofthose bacteria that have escaped (Fig. S4D). Conversely, upon infection with osteo-blasts, some LAC-GFP bacteria were killed and the death of the host cells was alsovisualized (Fig. S5A and B and Video S4). The decrease in and high SD of both the M1and M2 coefficients from 4 to 8 h p.i. are indicative of both a decrease in GFP expression(death of bacteria) and a decrease in colocalization (death of host cell) (Fig. S5C).Together, the time-lapse experiments reveal a difference in sensitivity of fibroblasts andosteoblasts to bacterial infection in regard to microbicidal activity and death.

S. epidermidis persists during localization within acidic phagosomes. Despitebeing unable to escape the phagolysosome, S. epidermidis is able to persist in host cells.

FIG 4 S. epidermidis does not escape the phagolysosome by 6 h. Host cells were infected withFITC-labeled S. aureus 6850, FITC-labeled S. epidermidis RP62A, S. aureus LAC-GFP, or S. epidermidis1457-GFP at a multiplicity of infection of 50. The difference in arbitrary fluorescence units (ΔAFU) in thepresence and absence of monensin, normalized to the invasion rate {ΔAFU � [(AFU�monensin �AFU�monensin)/AFU�monensin] � 105}, was used as the readout for pH. Larger negative values correspondto more-acidic (i.e., lower) pH values. Staphylococci localization in infected MRC5 (human fibroblast) (A),NHOst (human osteoblast) (B), and MC3T3 (mouse osteoblast) (C) cells is shown. Data are mean values(�standard deviations of the means) from two independent experiments done in triplicate. *, P � 0.05,**, P � 0.01, ***, P � 0.001, ****, P � 0.0001. Significance indicated near strain designations was gatheredfrom a comparison of results at 2 and 6 h p.i.; significance indicated near data points was gathered froma comparison of results at 6 h p.i. between the corresponding S. aureus and S. epidermidis strains.

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We next assessed peribacterial pH to validate that bacteria are located within acidicvesicles and to determine whether S. epidermidis modulates the pH of phagolysosomesduring intracellular persistence. We measured peribacterial pH during host cell infec-tion with live or heat-killed (HK) bacteria labeled with Oregon Green 488, as describedby Porte et al. (69). HK bacteria were used as a reference to confirm the pH within thephagolysosomes. By 2 h p.i., phagosomes containing live and HK bacteria were at anaverage pH of 4.8 for each of the host cells (Fig. 7). Treating host cells with monensinprior to infection resulted in phagosomes with an average pH of 6.6. There was littlechange in the peribacterial pH between 3 and 15 h p.i. for S. epidermidis RP62A and HKbacteria. S. aureus 6850 was the only bacterium tested that reached an average pH of5.7, 5.9, and 5.3 for human fibroblasts and human and mouse osteoblasts, respectively.These findings suggest that during intracellular infection, S. epidermidis persists for atleast 15 h in an acidic environment, whereas S. aureus 6850 either modulates thephagosomal pH, translocates into the cytosol, or both. In addition, these data suggestthat a direct mechanism to escape the phagosome or modulate the acidic environmentis not required to persist intracellularly.

DISCUSSION

It is widely accepted that biofilm formation is a major reason for the establishmentand persistence of S. epidermidis infections (10, 24, 25). We show here that intracellularpersistence likely also plays a role in S. epidermidis pathogenesis. Multiple clinical S.epidermidis isolates were able to persist for several days in nonprofessional phagocytesfound at the site of joint arthroplasty, localized in acidic phagolysosomes. We did notobserve S. epidermidis escape the phagolysosome; however, it was able to persist in thephagolysosome, outlive its host, and later escape to the extracellular compartmentupon host cell death. It is possible that this contributes to the typical delayed onset of

FIG 5 S. epidermidis bacteria do not escape phagolysosomal compartments. Live MRC5 (human fibro-blast) cells were infected with S. epidermidis 1457-GFP at a multiplicity of infection of 50 and visualizedby confocal microscopy. Nuclei, blue; 1457-GFP, green; lysosomes, magenta; cell membrane, cyan. (A)Image of human fibroblasts 7.5 h postinfection (p.i.). The dotted circle in the merged image representsthe area analyzed for colocalization. (B) Quantification of the Manders’ colocalization coefficient M1(fraction of phagolysosomal marker in colocalization with bacteria) or M2 (fraction of bacteria incolocalization with phagolysosomal markers) at 7.5 h p.i.

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symptoms associated with S. epidermidis infections such as PJI. In addition, we havepreviously shown and confirmed in this study that both acidic environments andintracellular persistence promote the formation of S. epidermidis SCVs, which maycontribute to antibiotic tolerance and chronic infection. The results of our studysuggest that fibroblasts, osteoblasts, and possibly other nonprofessional phagocytesconstitute an intracellular niche potentially underlying some reported relapses offoreign-body-associated infections after foreign body removal and antibiotic therapy.

We have previously shown that S. epidermidis can be cultured from bone approxi-mately 7 weeks after infection in rats (70) and rabbits (71), using a foreign bodyosteomyelitis model. In addition, Kwakman et al. (72) showed that S. epidermidis canpersist in peri-implant tissue from an experimental catheter-associated infection modelin mice. However, the method of culture of the tissues for each of these studies doesnot distinguish between intracellular and extracellular bacteria. Here, we found that S.epidermidis is capable of invading osteoblasts and fibroblasts in vitro. These findingscorroborate previous reports which demonstrate that S. epidermidis can invade humanbone cells after 2 h (48). We also demonstrated that S. epidermidis can persist intracel-lularly for longer than a week and that agr and PSM expression is not required for eitherinvasion or persistence. Another difference between our studies and those previouslyperformed is that we found S. epidermidis 1457 capable of invading and persisting inprimary human osteoblasts, whereas Khalil et al. demonstrated this strain to be unableto invade the MG63 bone cell line (48). This difference may be due to a number offactors but is most likely due to a difference in cell type tested. This is supported by our

FIG 6 S. epidermidis escapes to the extracellular environment upon eukaryotic host cell death. Live NHOst(human osteoblast) cells were infected with S. epidermidis 1457-GFP at a multiplicity of infection of 50and visualized by confocal microscopy. Nuclei, blue; 1457-GFP, green; lysosomes, magenta; cell mem-brane, cyan. (A and B) Images of infected human osteoblasts 3 h postinfection (p.i.) (A) and 5.5 h p.i. (B).The dotted circles represent the areas zoomed in for visualization of colocalization shown in the top rightcorners of the merged images. (C) Quantification of the Manders’ colocalization coefficient M1 (fractionof phagolysosomal marker in colocalization with bacteria) or M2 (fraction of bacteria in colocalizationwith phagolysosomal markers) at 3 h p.i.

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findings (Fig. 1A) and those of others showing that invasion is strain and host cell typedependent (73). Previously, it has been shown that S. aureus invasion is dependent onthe �5�1 integrin, whereas this receptor is not required for S. epidermidis strain 19invasion of bone cells (48). The mechanism of invasion observed in osteoblasts andfibroblasts in this study is not currently known. Possibly, invasion can be accidental,where bacteria may be inadvertently phagocytosed during the wound healing process,or invasion can be accomplished via binding to a specific receptor, currently unknown,allowing for endocytosis. Future experiments are needed to understand how S. epider-midis is taken up into these cells. Our results also show little eukaryotic cell death overthe course of infection, suggesting that human fibroblasts and osteoblasts (albeit to aslightly lower extent) are able to tolerate intracellular infection with S. epidermidis. Wealso show osteoblasts to have microbicidal activity. Differences in this activity betweenthe human and mouse osteoblasts are likely due to species differences and perhaps adifference in function between primary cells and cell lines. In addition, our resultsobtained from intracellular infection in human and mouse osteoblasts reveal a differentfate between isolates associated with sepsis or PJI, with sepsis-associated strains ableto persist at stable levels. Together, these results show that the expression of virulencefactors, such as agr and PSMs, is not required for invasion and intracellular persistencegenerally but perhaps plays a role in the ability to persist in microbicidal host cells. Thishypothesis is supported by the work of Cheung et al., who previously showed thatisogenic sepA and aps mutants of strain 1457 had a reduced ability to survive afterphagocytic interaction with human neutrophils in comparison to the wild-type strain(31).

FIG 7 Intracellularly persisting S. epidermidis bacteria are located within an acidic environment. Perib-acterial pH was evaluated in MRC5 (human fibroblast) (A), NHOst (human osteoblast) (B), and MC3T3(mouse osteoblast) (C) cells treated with or without monensin and infected with unlabeled or OregonGreen 488-labeled bacteria (multiplicity of infection of 100). At 2 and 15 h postinfection (p.i.), fluores-cence was measured with a spectrofluorometer with excitation at 498 nm and 450 nm and emission at520 nm. Periphagosomal pH was calculated by comparing the 498 nm/450 nm ratio of each sample witha standard pH titration curve. Data are mean values (�standard deviations of the means) from twoindependent experiments. NT, not treated.

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It has been reported that there are differences in gene expression between free-floating and adherent bacterial cells. For example, in S. aureus planktonic cells, agr andgenes encoding toxins are upregulated, while no toxin gene was expressed at highlevels in the S. aureus biofilm cells (50). In addition to changes in metabolism, biofilmcells also had an increase in the expression of ica genes in comparison to that ofplanktonic bacteria (50). Overall, it has been suggested that planktonic bacteria ex-pressing toxins are generally more virulent and have a greater ability to cause acuteinfection than biofilm cells, whereas biofilm cells tend to employ defensive molecularmechanisms to promote their survival, at times resulting in chronic infection (50, 74,75). Here we compared the invasion and persistence of one chronic-infection and oneacute-infection S. aureus PJI isolate and showed that both isolates are successfully ableto invade and persist within mouse osteoblasts. As seen for the S. epidermidis strains,the expression of virulence factors may not be required for invasion. However, furthercharacterization of these two isolates (and other chronic and acute infection-associatedisolates) and further time points are needed to distinguish any changes in the ability topersist past 7 days and to identify genes responsible for this persistence.

SCV formation is induced in response to diverse environmental pressures (51–53).SCVs typically comprise a minor proportion of the source population and are commonin chronic infections (54–58). S. epidermidis SCVs are common in PJI (8). Here weshowed there to be no difference in ability to invade cells between pairs of an isogenicSCV or closely related SCV and an NCP S. epidermidis isolate (see Fig. S2E and F in thesupplemental material), as previously shown for S. aureus (65). In addition, we showeda positive correlation between SCV formation and time of intracellular persistence inhuman fibroblasts with multiple staphylococcal strains and extended these findings toinfection in osteoblasts. Previously, we have shown that the number of SCVs can bereduced by treatment with lysosomotropic alkalinizing agents. For at least the firstweek, SCVs do not significantly contribute to survival, as there is no change in the totalnumber of CFU recovered from cells treated with these drugs (49). Although theincreasing numbers of SCVs suggest adaptation to the acidic environment, there maybe another role this subpopulation plays during infection. Recently, two groups haveimplicated SCVs in proinflammatory cytokine suppression. Ou et al. showed that after24 h, intracellular infection with NCP S. aureus in a human bronchial epithelial cell lineinduced a robust proinflammatory response evidenced by increased Toll-like receptor2 (TLR2), interleukin 1 (IL-1), IL-6, and IL-12 expression, whereas SCV infection inducedonly TLR2 expression (65). Magrys et al. showed that 4 h after infection of THP-1 cellswith S. epidermidis SCVs isolated from a hip PJI, there was an increase in AKT phos-phorylation compared to that in NCP-infected and noninfected cells (76). This group didnot show direct evidence for proinflammatory cytokine suppression, but because thephosphatidylinositol 3-kinase (PI3K)-AKT pathway has been shown to be involved withcytokine suppression, they suggested this role. Thus, together with our findings, thissuggests that intracellular persistence and tolerance to this persistence are maintainedby SCVs via mechanisms currently unknown, possibly by modulating signaling path-ways.

Classified as nonprofessional phagocytes, both fibroblasts and osteoblasts usemechanisms similar to those used by macrophages to phagocytose and degrade thecontents within their phagosomes (77–79). Using live time-lapse fluorescence micros-copy, we showed that within 2 h of infection, bacteria are taken up by osteoblasts andfibroblasts into acidic phagosomes, where some but not all S. epidermidis cells are killed.In addition, our results indicate that S. epidermidis is unable to escape the phagosomesor modulate the acidic environment after 15 h. This is different from results generatedwith S. aureus, both in our study and in those by others, describing phagosomal escapeand replication in the cytosol (15, 40, 66, 80). However, as not all S. aureus isolates areable to escape and some escape at later time points, our findings do not definitivelyprove that S. epidermidis cannot escape the phagolysosome. Because phagosomalescape for S. aureus is dependent upon PSM� expression (66), whose homologue isexpressed in much lower concentrations by S. epidermidis (31), it is plausible that this

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organism simply requires more time for phagosomal escape than does S. aureus. Mostimportantly, we were able to show that viable S. epidermidis is capable of escaping intothe extracellular environment upon host cell death. Together, the results suggest thatS. epidermidis is able to tolerate the acidic phagosomal environment for several days.

In conclusion, our findings show that intracellular infection of osteoblasts, fibro-blasts, and possibly other nonprofessional phagocytes comprise a second and equallyimportant mechanism, beyond biofilm formation, enabling evasion from host immunedefense and permitting bacteria to persist during infection and possibly treatment.Given that current treatments may not kill intracellularly persisting bacteria, targetingthis population may contribute to increased treatment success. Additionally, SCVformation may contribute to the subtle and silent nature of infection through as-yet-undescribed processes.

MATERIALS AND METHODSMicroorganisms. Bacterial strains used in this study are detailed in Table S1 in the supplemental

material. Prior to assays, staphylococci were grown overnight in 10 ml of tryptic soy broth (TSB) at 37°Cwith rotation. Bacteria expressing superfolder green fluorescent protein (sGFP) were grown in 10 ml TSBsupplemented with 10 �g/ml chloramphenicol. Bacteria were harvested by centrifugation, washed twicewith phosphate-buffered saline (PBS), and resuspended in assay medium. Bacterial cell numbers wereestimated spectrophotometrically at 600 nm and/or using McFarland standards. Heat-killed (HK) bacteriawere prepared by heating the bacteria for 10 to 15 min at 65°C. Lack of viability was confirmed by nogrowth after plating (data not shown). Prior to experimentation, S. epidermidis and S. aureus stockcultures were confirmed for agr expression via the production of PSM� and/or PSM�2 via reversetranscription PCR or ELISA (data not shown). SCVs and NCP isolates from the same joint were previouslyassessed for relatedness by pulsed-field gel electrophoresis (PFGE), as described previously (81). IDRL-8933 and IDRL-8934 had a 3-band difference, whereas IDRL-8864 and IRL-8866 were indistinguishable(81). To confirm that both SCV and NCP isolates originated from the same clone, whole-genomesequencing (WGS) had been previously conducted at the Department of Infection Control Science atJuntendo University Graduate School of Medicine (Tokyo, Japan) (82). WGS results were insufficient toconfirm that IDRL-8933 and IDRL-8934 were clonally related. However, WGS revealed approximately 10mutations between IDRL-8866 and IDRL-8864, supporting the theory that these two strains are clonal(82).

Cell culture. Human lung fibroblasts (MRC5 cells) were maintained in RPMI 1640, supplemented with10% heat-inactivated fetal bovine serum (Sigma). Murine osteoblasts (MC3T3-E1) were maintained inminimum essential medium alpha (MEM�) supplemented with 10% heat-inactivated fetal bovine serum.Mouse osteoblast cell authentication was performed by IDEXX BioResearch using short tandem repeat(STR) DNA profiling. Primary normal human osteoblast (NHOst) cells were maintained in osteoblast basalmedium supplemented with OGM SingleQuots (Lonza). Human osteoblast cell authentication wasperformed by the Mayo Clinic Genotyping Core using STR DNA profiling. Osteoblasts were alsoconfirmed by alkaline phosphatase and bone mineralization staining (data not shown).

Intracellular persistence. (i) Lysostaphin-daptomycin protection assay. Host cells were infectedat a multiplicity of infection (MOI) of 25 for intracellular assay. Host cells were washed daily with PBS andlysostaphin (Sigma) or daptomycin (to kill extracellular bacteria) and provided fresh medium supple-mented with 10% fetal bovine serum. At 0, 3, 5, and 7 days p.i., host cells were lysed and serial 10-folddilutions of the cell lysates were plated onto sheep blood agar (SBA) to detect and quantitate intracellularbacteria. CFU were enumerated after overnight incubation at 37°C in air. The SCV phenotype wasdetermined by size after a second overnight incubation at room temperature. Only intracellularlypersisting bacteria were monitored over time, as no colonies were found in the washing medium afterthe lysostaphin/daptomycin treatment (data not shown).

(ii) Viability. Host cells were detached from wells with trypsin-EDTA (0.25%) (Thermo Fisher), and a100-�l aliquot was diluted 1:1 in 0.4% trypan blue solution. The percentage of live cells was calculatedusing a Countess II FL automated cell counter (Thermo Fisher); two independent samples were analyzedper time point.

Phagolysosomal localization. (i) Confocal microscopy of fixed cells. S. epidermidis RP62A wasgrown and harvested as described above. Bacterial cells were washed and stained with 5 mM CFSE(eBioscience) in the dark for 10 min at room temperature. Host cells grown on glass coverslips were theninfected at an MOI of 50 with the CFSE-labeled bacteria for 2 h at 37°C in 5% CO2. Following the 2-hincubation, host cells were washed and incubated with 20 �g/ml lysostaphin to kill extracellular adherentbacteria. Host cells were then further incubated for 1 h at 37°C in 5% CO2. The cells were washed, fixed,and permeabilized. After blocking, the cells were incubated with primary antibodies overnight at 4°C,followed by three washes with PBS and incubation with secondary antibodies for 1 h and then threemore washes. Antibodies to human lysosomal-associated membrane protein-2 (LAMP-2) (H4B4; Abcam)were used at 1:100. LAMP-2 is abundantly and ubiquitously expressed on lysosomal membranes and isused to mark mature endosomes and lysosomes. The fluorophore-conjugated donkey anti-mousesecondary antibody was used at 1:500. Confocal microscopy was performed using an LSM 780 micro-scope (Carl Zeiss, Germany) with a 40� objective.

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(ii) Confocal microscopy of live cells. S. epidermidis 1457 expressing sGFP was grown and harvestedas described above. Host cells were infected at an MOI of 50 for 1 h at 37°C in 5% CO2. Host cells werethen washed as described above, and a CytoPainter lysosomal staining kit (Abcam) was used to stain forlysosomes. The lysotropic indicator from the kit is designed to selectively accumulate in the lysosomesof live cells. Cells were then incubated for 30 min at 37°C in 5% CO2, washed, and stained with NucBlue(Invitrogen) for 5 min, followed by staining with the CellMask deep red plasma membrane stain (ThermoFisher) for 5 to 10 min, both at 37°C in 5% CO2. The cells were washed, and phenol-red free medium wasadded. Confocal microscopy was performed with an LSM 780 microscope (Carl Zeiss, Germany) using a40� objective in a controlled chamber providing incubation at 37°C in 5% CO2.

(iii) Colocalization. Colocalization of bacteria and phagolysosomes was determined by selecting atleast 15 randomly chosen regions of interest (ROI) containing intracellular bacteria using ImageJ Coloc2 software to determine the Manders’ coefficients. There is one Manders’ coefficient per channel, andeach coefficient is proportional to the amount of fluorescence in that channel that colocalizes with thefluorescence in the other channel over its total intensity. The coefficient varies from 0 to 1, with 0indicating no colocalization and 1 indicating perfect colocalization (83). The colocalization in livetime-lapse images was determined by averaging the coefficients of each image within respective timeframes.

(iv) Flow cytometry phagolysosome escape assay. The flow cytometry phagolysosome escapeassay, which monitors the pH of the environment of intracellular staphylococci, was performed asdescribed by Lâm et al. (40), with slight modification. Bacterial cultures were grown and harvested asdescribed above. Washed bacterial pellets were then resuspended in 0.1 M sodium bicarbonate buffer,and bacteria were labeled with fluorescein isothiocyanate (FITC) (Invitrogen) in the dark with rotation for30 min at room temperature. Bacterial cells were washed with Hanks balanced salt solution (HBSS) toremove unbound dye and then resuspended in HBSS. Prior to infection, host cells were seeded at 3 �105 cells per well and grown overnight in a 24-well culture disk to subconfluence. Host cells were placedon ice for 10 min to synchronize phagocytosis, and cells were then infected at an MOI of 50. Followingincubation on ice for 15 min, the cells were moved to 37°C in 5% CO2 for 55 min. The cells were washedand incubated with medium supplemented with lysostaphin (20 �g/ml) at 37°C for 30 min. The cellswere returned to the incubator for an additional 55 min or 4 h and 55 min, resulting in an overallinfection time of 2 or 6 h, respectively. Cells were washed with PBS, trypsinized, and transferred to 5-mlround-bottom tubes. Samples were incubated with or without 50 �M monensin for 20 min at 37°C,followed by propidium iodide treatment for live/dead staining. Samples were analyzed using FACSCantoX. For each cell type, a preset fixed gating and a fixed amplification were used. The invasion rate withmonensin (total internalized bacteria), expressed as numbers of arbitrary fluorescence units (AFU), wasdetermined according to the formula AFU � [(mean fluorescence intensity of cells in M) � (percentageof cells in M)]/1,000 and normalized to the mean fluorescence intensity of each corresponding bacterialpreparation. (The FITC-positive gate is denoted M.) The difference in AFU (ΔAFU) in the presence orabsence of monensin, normalized to the invasion rate {ΔAFU � [(AFU�monensin � AFU�monensin)/AFU�monensin] � 105}, was used as the readout for pH. Larger negative values correspond to more-acidic(i.e., lower) pH values.

Measurement of peribacterial pH. Live (overnight) or HK (prepared as described above) bacteriawere labeled with the pH reporter dye Oregon Green 488 (OG-488) (Invitrogen), which can assess pHvalues between 3.5 and 7 (69). The experimental setup for this assay was as described by Tranchemon-tagne et al. (18). Live and HK cultures were washed with Dulbecco’s phosphate-buffered saline (D-PBS),resuspended in OG-488 (10 mg/ml), and incubated for 30 min at 4°C in the dark. Labeled bacteria werecentrifuged, and the labeling reaction was stopped by adding Tris-HCl (pH 8.3) to a final concentrationof 100 mM. Bacteria were incubated at 4°C in the dark for 15 min, washed, and used for infection.Labeling with OG-488 did not affect the viability of live bacteria as measured by plating on bovine serumalbumin (BSA) (data not shown).

To determine the pH of the environment surrounding S. epidermidis, host cells were grown in phenolred-free RPMI 1640 in tissue culture-treated, black-walled 24-well microplates (PerkinElmer). Cells weretreated with or without 50 �M monensin and infected with Oregon Green 488-labeled or unlabeledbacteria at an MOI of 100, according to the procedure described above. At 2 and 15 h p.i., 0.1% trypanblue was added to infected cells for 1 min to quench the fluorescence of the remaining extracellularbacteria, and the cells were then washed three times with cell medium. With the emission wavelengthat 520 nm, the ratio of fluorescence with excitation at 498 to that at 450 nm was measured using aFluoroskan Ascent (Thermo Scientific). As described by Levitz et al. (84), a standard curve was generatedfor each condition by obtaining excitation ratios at 498 and 450 nm in infected cells where thephagosomal and extracellular pH values were equilibrated in nigericin buffers of defined pH (0.028 mMnigericin, 10 �M monensin, 150 mM sodium chloride, 80 mM potassium chloride, 1 mM magnesiumchloride, 5.5 mM glucose, 2 mM calcium chloride, and 10 mM HEPES) (data not shown). Backgroundvalues for excitation at 498 and 450 nm with emission at 520 nm for cells infected with unlabeledbacteria were subtracted from readings obtained with labeled bacteria. To calculate the averagephagosomal pH of the samples, the ratios of excitation at 498 to that at 450 nm were compared with thestandard pH titration curve generated from nigericin-permeabilized cells. HK bacteria were used as areference to confirm the pH over time in the phagolysosomes, as dead bacteria would not activelymodulate or escape the phagolysosome.

Statistical methods. Statistical analysis was performed using a two-tailed t test. We used an alphavalue of 0.05, so P values of �0.05 were considered significant (*, P � 0.05, **, P � 0.01, ***, P � 0.001,and ****, P � 0.0001). All error bars represent standard deviations (SD).

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SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/IAI.00237-18.

SUPPLEMENTAL FILE 1, PDF file, 0.6 MB.SUPPLEMENTAL FILE 2, AVI file, 13.5 MB.SUPPLEMENTAL FILE 3, AVI file, 6.0 MB.SUPPLEMENTAL FILE 4, AVI file, 18.8 MB.SUPPLEMENTAL FILE 5, AVI file, 15.8 MB.

ACKNOWLEDGMENTSS. aureus strain 6850 was kindly provided by Bettina Löffler (Institute of Medical

Microbiology, Jena, Germany). S. epidermidis strains 1457, 1457ΔPSM�, Tü3298, andTüF38 were kindly provided by Michael Otto (NIAID NIH, Bethesda, MD). S. epidermidisstrain 1457 expressing GFP was kindly provided by Alexander Horswill (University ofColorado Department of Immunology and Microbiology, Denver, CO). MRC5 cells werekindly provided by Kelli Black (Mayo Clinic Virology/Parasitology Laboratory, Rochester,MN), and MC3T3 cells were kindly provided by Jennifer Westendorf (Mayo ClinicDepartment of Orthopedic Surgery, Rochester, MN). Primary normal human osteoblastswere kindly provided by Paul German Norambuena Morales (Mayo Clinic Departmentof Orthopedic Surgery, Rochester, MN).

This work was supported by NIH grant GM055252, a Ph.D. Training Grant in BasicImmunology (T32 AI07425-21), NIH grant R01 AR056647, and NIH grant R01 AI91594.

K.P. performed the experiments. R.P. supervised K.P. and helped edit and revise themanuscript.

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