Antimicrobial susceptibility and biofilm formation of Staphylococcus epidermidis small colony variants associated with prosthetic joint infection

Diagnostic Microbiology and Infectious Disease 74 (2012) 224–229

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Diagnostic Microbiology and Infectious Disease j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d i a g m i c r o b i o

Bacteriology

Antimicrobial susceptibility and biofilm formation of Staphylococcus epidermidis small colony variants associated with prosthetic joint infection☆ Awele N. Maduka-Ezeh a, Kerryl E. Greenwood-Quaintance b, Melissa J. Karau b, Elie F. Berbari a, Douglas R. Osmon a, Arlen D. Hanssen c, James M. Steckelberg a, Robin Patel a, b,⁎ a b c

Division of Infectious Diseases, Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905, USA Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN 55905, USA

a r t i c l e

i n f o

Article history: Received 27 February 2012 Accepted 29 June 2012 Available online 15 August 2012 Keywords: Small colony variant Staphylococcus epidermidis

a b s t r a c t We determined the frequency of isolation of non-aureus staphylococcal small colony variants (SCVs) from 31 patients with staphylococcal prosthetic joint infection (PJI) and described the antimicrobial susceptibility, auxotrophy, and biofilm-forming capacity of these SCVs. Eleven non-aureus SCVs were recovered, all of which were Staphylococcus epidermidis, and none of which was auxotrophic for hemin, menadione, or thymidine. Aminoglycoside resistance was detected in 5. Two were proficient, and 7 were poor, biofilm formers. With passage on antimicrobial free media, we observed a fluctuating phenotype in 3 isolates. We also noted a difference in antimicrobial susceptibility of different morphology isolates recovered from the same joints despite similar pulsed-field gel electrophoresis patterns. Our findings suggest S. epidermidis SCVs are common in PJI, and while they have a similar appearance to S. aureus SCVs, they do not necessarily share such characteristics as aminoglycoside resistance; auxotrophy for hemin, menadione, or thymidine; or enhanced biofilm formation. We also underscore the importance of antimicrobial susceptibility testing of all morphologies of isolates recovered from PJI. © 2012 Elsevier Inc. All rights reserved.

1. Introduction The numbers of hip arthroplasties performed in the United States rose from 138,440 in 1990 to 226,615 in 2004 (Kurtz et al., 2008). With the increased number of prosthetic joint placements, there was an increase in the number of associated infections over the same period (Kurtz et al., 2008). Prosthetic joint infection (PJI) has significant consequences for patients in terms of quality of life and economic impact on hospitals, payers, and patients (Bozic and Ries, 2005; Sculco, 1995). Staphylococci are the most common etiologic agents of PJI (Del Pozo and Patel, 2009; Geipel, 2009). Apart from PJI, staphylococcal infection of other orthopedic implantable hardware, including spinal fusion devices and limb fixation devices, causes significant morbidity (Labbe et al., 2003; Mok et al., 2009). Some cases of persistent, difficult-to-treat PJI are associated with a naturally occurring population of organisms which exhibits physiologic, biochemical, and colonial morphologies different from usual isolates (Proctor et al., 2006; von Eiff et al., 2000). These variants are called small colony variants (SCVs), due to their morphology on solid ☆ Presented in part at the Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, September 17–20, 2011. ⁎ Corresponding author. Tel.: +1-507-538-0579; fax: +1-507-284-4272. E-mail address: [email protected] (R. Patel). 0732-8893/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2012.06.029

growth media. In addition to causing PJI, SCVs have been described as causing infection associated with other prosthetic material, including cardiac devices (Maduka-Ezeh et al., 2012). Infections caused by SCVs may pose a multilevel challenge in terms of identification and management. Difficulties are encountered in identifying SCVs in the clinical microbiology laboratory. On culture media, SCVs grow slowly and form pinpoint colonies such that they may be overlooked or overgrown by wild-type colonies in cases of dual infection. SCVs may demonstrate reduced hemolysis on blood agar. Staphylococcus aureus SCVs demonstrate variable results on coagulase testing as well as diminished pigment production (Proctor et al., 2006; von Eiff, 2008), which may result in misidentification. Even when correctly identified, there may be difficulty in antimicrobial susceptibility testing due to a slow growth rate (von Eiff, 2008). Staphylococcal SCVs described to date have primarily been S. aureus. S. aureus SCVs have been described as being auxotrophic for certain nutrients, typically hemin, menadione, or thymidine (Lannergard et al., 2008; Norstrom et al., 2007). Hemin or menadione auxotrophy is associated with defects in electron transport and, consequently, altered membrane potential (Baumert et al., 2002; McNamara and Proctor, 2000). The abnormal membrane potential may confer on these variants innate resistance to antibiotics such as aminoglycosides, which depend on membrane potential to gain access to intracellular target sites (Baumert et al., 2002). Relative resistance to other antibiotics, including daptomycin, vancomycin,

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and fusidic acid, has also been described (Norstrom et al., 2007; Wu et al., 2009). Resistance to folate antagonists such as trimethoprim– sulfamethoxazole (TMP-SMX) has been noted in thymidine auxotrophic SCVs which may bypass the effect of these antimicrobials by uptake of environmental thymidine (Besier et al., 2008; Zander et al., 2008). It has been suggested that antibiotics (systemic or, in the case of PJI, local) may actually select for SCVs (Bayston et al., 2007; Proctor et al., 2006). Using transmission electron microscopy, Wellinghausen et al. (2009) found enterococcal SCVs to have empty “ghost” cells, thick cell walls, heterogenous sizes, and aberrant shapes compared to the normal counterparts. Recent studies have demonstrated the enhanced biofilm-forming ability of laboratory-derived SCVs of S. aureus compared to normal morphology counterparts (Singh et al., 2009, Singh et al., 2010). Another characteristic thought to play a role in the persistence of the SCVs is intracellular existence, providing protection from the host immune system and antimicrobial agents (Darouiche and Hamill, 1994; Lannergard et al., 2008; Norstrom et al., 2007). S. aureus SCVs may persist intracellularly for prolonged periods because, unlike wildtype parent strains, they are less toxic to the cells they infect and thus induce slower cell death (von Eiff et al., 1997; von Eiff et al., 2000). It has been postulated that under antibiotic pressure, wild-type strains convert to SCVs and become intracellular, protecting themselves from antimicrobials and causing persistent low-grade (even subclinical) infections (Brouillette et al., 2004; von Eiff et al., 1997; Zander et al., 2008). At the completion of antimicrobial therapy, SCVs may provide a source for relapse of infection, emerging to cause overt disease (Proctor et al., 1995). Most of the available literature regarding SCVs deals with clinical and laboratory-derived strains of S. aureus. Whether or not the above description applies to non-aureus staphylococcal SCVs is unknown. While there have been descriptions of S. aureus SCVs causing native and prosthetic orthopedic infections (Proctor et al., 1995; Sendi et al., 2006), the prevalence of non-aureus staphylococcal SCVs in clinical infections in general, and in orthopedic infections in particular, is largely unknown. We determined the relative frequency of isolation of non-aureus staphylococcal SCVs, the associated species, and the characteristics, including auxotrophy, antimicrobial susceptibility, biofilm-forming capacity, and rate of reversion to normal colony morphology.

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2.2. Organism identification Organisms were identified using Gram staining, catalase, and coagulase testing. Explant sonicate culture plates were examined for SCVs, defined as pinpoint colonies, 1/10th or less the size of normal staphylococcal colonies when grown on sheep blood agar (Becton Dickinson, Franklin Lakes, NJ, USA) (Proctor and Peters, 1998; Proctor et al., 2006). All staphylococci were archived at −70 °C. Staphylococci that tested coagulase negative, as well as all SCVs (coagulase positive or negative), were identified to the species level by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (Bruker Daltonics, Billerica, MA, USA) as previously described (Alatoom et al., 2011). Thereafter, no further studies were performed on S. aureus isolates. Partial 16S ribosomal RNA gene sequencing was also performed on coagulase-negative staphylococcal SCVs (Alatoom et al., 2011). 2.3. Antimicrobial susceptibility testing Agar dilution and disc diffusion susceptibility tests were done on non-aureus SCVs per Clinical laboratory and Standards Institute (CLSI) guidelines (CLSI, 2012) and Etest (bioMérieux, Marcy L'Etoile, France) per manufacturer guidelines except that, in addition to reading susceptibilities at 20–24 h, they were read at 48 h (to accommodate the slow growth rate of SCVs) (Proctor et al., 1995). Susceptibilities at both time points were considered valid if the MIC of the control strain remained within the CLSI-specified quality control range. mecA polymerase chain reaction (PCR) was done as previously described (Kohner et al., 1999). 2.4. Pulsed-field gel electrophoresis testing For SCVs that had normal colony morphology organisms isolated from the same joint, relatedness between the SCV and the normal morphology isolate was assessed by pulsed-field gel electrophoresis (PFGE) using SmaI. SCV-normal morphology pairs were considered clonally related if they were indistinguishable (no band difference) or closely related (2- to 3-band difference) by PFGE (Tenover et al., 1995). Only clonally related pairs were compared in subsequent “pair” studies. 2.5. Auxotrophy testing

2. Materials and methods From March 2010 to March 2011, isolates were obtained from individuals who met criteria for PJI (sinus tract communicating with prosthesis, gross purulence noted at surgery, and/or acute inflammation on intraoperative frozen section histology) (Del Pozo and Patel, 2009; Piper et al., 2009), who had explanted prosthetic joints sent for sonication culture (Piper et al., 2009; Trampuz et al., 2007), and whose explant cultures grew ≥20 CFU/10 mL sonicate fluid of a Staphylococcus species (without concurrent growth of a nonstaphylococcal species).

2.1. Bacterial strains ATCC 29213 and 25923 were used as controls for agar dilution and disc diffusion susceptibility testing, respectively (CLSI, 2012). RP62A (ATCC 35984), a well-characterized biofilm-forming S. epidermidis strain, was used as a control for biofilm formation experiments (Frank et al., 2007). S. aureus menD and hemB mutants (von Eiff et al., 2006), kindly provided by Dr Christof von Eiff (Department of Medical Microbiology, University of Münster Medical School, Münster, Germany), were used as controls for assessing auxotrophy.

Auxotrophy for menadione, thymidine, and hemin was evaluated on non-aureus SCV isolates using agar and disc methods. For the agar method, following overnight growth on sheep blood agar at 37 °C in air, 3–4 similar colonies were inoculated onto a Mueller Hinton agar (MHA) plate and onto 3 additional MHA plates, 1 each of which was supplemented with 10, 25, and 125 μg/mL of hemin, menadione, and thymidine, respectively. The plates were incubated for 24 h at 37 °C in air. For the disc method, overnight cultures were diluted to a 0.5 McFarland standard and inoculated onto a MHA plate. Discs impregnated with menadione (25 μg/mL) or thymidine (100 μg/mL) were placed on the inoculated plates (to assess menadione and thymidine auxotrophy, respectively). Hemin auxotrophy was tested using commercially available hemin (X-factor) discs (Sigma-Aldrich, St Louis, MO, USA). Isolates were considered auxotrophic if they demonstrated normal colony growth on the supplemented versus the nonsupplemented MHA plate and/or if they showed increased growth around the impregnated disc compared to the periphery. CO2 auxotrophy testing was done by simultaneously passaging isolates in air and CO2. Each passaging step was performed in duplicate. Isolates were considered CO2 auxotrophs if they grew as normal-sized colonies on the plates incubated in CO2 and as SCVs on the plates incubated in air.

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2.6. Effect of passaging The effect of serial passage of non-aureus SCVs on antimicrobial free media was examined. The isolates were taken from −70 °C and grown at 37 °C in air on sheep blood agar plates. Isolates that showed no growth of SCVs at this point were not passaged further. After overnight growth, each SCV isolate was subcultured onto 2 fresh sheep blood agar plates and again incubated at 37 °C in air for 24 h. Plates were then examined for the presence and proportion of normal-sized colonies to SCV. This was continued for at least 8 passages per isolate, tested in duplicate. 2.7. Biofilm growth measurement A 96-well polystyrene microtiter plate assay was used to assess biofilm formation of the non-aureus SCVs, as previously described (Deighton and Borland, 1993; Kristich et al., 2004), with minor modifications. Briefly, for each isolate, cultures were adjusted with trypticase soy broth (TSB) to match the turbidity of a 1.0 McFarland standard and then diluted 1:50 in TSB. Two hundred μL aliquots were placed into 4 wells each of five 96-well microtiter plates (Corning Incorporated, Corning, NY) corresponding to incubation times of 6, 12, 24, 48, and 72 h. Cell growth was measured by OD600 on a Multiskan microtiter plate reader (Thermo Electron, Waltham, MA, USA). The culture medium was then discarded and wells washed twice by submerging plates in deionized water to remove nonadherent cells. Plates were air dried overnight. Biofilms were then stained with 0.1% safranin for 1 min, rinsed under running tap water to remove excess stain, and air dried. Stained biofilms were resuspended in 200 μL of 30% glacial acetic acid, and the OD492 measured. Wells containing uninoculated TSB were used as negative controls and S. epidermidis RP62A as a positive control. The biofilm index (mean biofilm OD492/mean planktonic OD600), which measures biofilm-forming capacity corrected for differences in growth rate (Deighton and Borland, 1993; Frank et al., 2007; Kristich et al., 2004), was calculated for each isolate at each time point. Differences in biofilm indices of isolates were assessed using the paired Student's t test with a P value of b0.05 being considered significant. 2.8. Transmission electron microscopy After overnight growth on sheep blood agar, isolates were submitted for transmission electron microscopy (TEM). Briefly, colonies were suspended in Trump's fixative (1% glutaraldehyde and 4% formalin in phosphate buffer) and centrifuged. The pellets were fixed in 1% glutaraldehyde, postfixed in 1% osmium tetroxide, and embedded in epoxy resin. Thick and ultrathin sections were cut on the cell pellets using an ultramicrotome. The ultrathin sections were mounted on copper grids, differentially stained using lead citrate, and examined on a JEOL-1400 transmission electron microscope (JEOL, Tokyo, Japan), operating at 80 kV. One SCV-normal pair (IDRL-8866 and IDRL-8864) indistinguishable by PFGE and 1 closely related pair (IDRL-8934 and IDRL-8933) were studied by electron microscopy. We used IDRL-8699, a thymidine auxotrophic S. aureus SCV (Maduka-Ezeh et al., 2012), for comparison as well as S. aureus ATCC 29213, a normal morphology S. aureus strain. 3. Results Over a 12-month period, 32 joints from 31 patients met enrollment criteria. (One patient had 2 joints explanted on different days.) The joint types included 20 prosthetic knees, 6 prosthetic hips, 2 prosthetic elbows, 2 spinal implants, and 1 each of wrist and shoulder prostheses. Eleven of the prostheses grew S. aureus, and 21

grew coagulase-negative staphylococci. SCVs were present in 3 of the S. aureus and 12 of the coagulase-negative staphylococcal PJI cases. No further testing was done on the S. aureus isolates. Of the 12 joints from which coagulase-negative SCVs were recovered, 6 also yielded normal morphology phenotypes. PFGE comparison of co-isolated non-aureus SCVs and normal colony isolates revealed 1 indistinguishable pair, i.e., 0-band difference: IDRL-8866 (SCV) and IDRL-8864 (normal morphology). There were 2 SCV/normal pairs (IDRL-8850/IDRL-8849 and IDRL-8934/IDRL-8933) that were closely related (i.e., having 2- to 3band difference) by PFGE. One SCV/normal morphology pair (IDRL8944/IDRL-8943) was possibly related (4-band difference) by PFGE. For the 2 remaining pairs, PFGE testing was not done because SCVs did not regrow on subculture from −70 °C. Joints from 6 patients yielded SCVs without accompanying normal morphology isolates. IDRL-8748, IDRL-8890, IDRL-8937, IDRL-8963, and IDRL-9048 were isolated from 5 patients. From the sixth patient, 2 distinct SCVs were isolated: IDRL-8826 appeared as pinpoint clear colonies and IDRL-8873 formed pinpoint white colonies. PFGE patterns of these co-isolated SCVs were indistinguishable; however, antimicrobial susceptibility patterns were different (Table 1), and so these SCVs were tested separately as distinct SCVs. In total, 11 SCVs were available for further study; all were S. epidermidis by MALDI-TOF mass spectrometry and partial 16S ribosomal RNA gene sequencing. 3.1. Antimicrobial susceptibility Antimicrobial susceptibility testing was done on the 11 S. epidermidis SCVs which retained the SCV morphology on regrowth from −70 °C and on the co-isolated normal morphology isolates for which PFGE demonstrated indistinguishable or closely related patterns to the co-isolated SCVs (Table 1). Of the 11 SCV isolates tested, 5 demonstrated resistance to at least 1 aminoglycoside tested (Table 2). Nine were mecA PCR positive; however, only 8 of the 9 demonstrated phenotypic oxacillin resistance. One SCV was TMP-SMX resistant (Tables 1 and 2). Of the 3 SCVs with co-isolated, clonally related normal colony morphology isolates, only 1 demonstrated less aminoglycoside susceptibility than its normal colony morphology counterpart (Table 1). The same SCV also demonstrated less TMP-SMX susceptibility than its normal colony morphology counterpart. The pair of different-appearing SCVs isolated from the same joint showed differences in aminoglycoside susceptibility despite having indistinguishable PFGE patterns (Table 1). 3.2. Auxotrophy testing Auxotrophy testing revealed 3 of the S. epidermidis isolates (IDRL8826, IDRL-8873, and IDRL-8934) to be CO2 auxotrophic. None was auxotrophic for hemin, menadione, or thymidine (Table 2). 3.3. Serial passaging With serial passage on antibiotic free media, 3 isolates (IDRL-8850, IDRL-8890, and IDRL-8963) were very stable, retaining the SCV morphology for more than 8 passages (Table 2). Three (IDRL-8748, IDRL-8866, IDRL-8937) were unstable, reverting to normal-sized (N1 mm) colonies after 3–6 passages and 5 (IDRL-8826, IDRL-8873, IDRL8934, IDRL-8944, IDRL-9048) were very unstable, reverting after 1–2 passages (Table 2). Three SCVs (IDRL-8826, IDRL-8866, IDRL-8934) demonstrated a “fluctuating phenotype” (Table 2): following reversion to normal-sized colonies, further passage of the normal-sized colonies resulted in the reappearance of the SCV phenotype. Two of these “fluctuating isolates” (IDRL-8826 and IDRL-8934) had shown initial reversion after 1–2 passages (very unstable) and 1 (IDRL-8866) after 3–6 passages (unstable).

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Table 1 Antimicrobial susceptibilitya of Staphylococcus epidermidis small colony variants. Isolate number

Gentamicin

Tobramycin

Kanamycin

Amikacin

Vancomycin

Trimethoprim– sulfamethoxazole

Oxacillin

Zone of inhibition around cefoxitin disk (mm)

mecA

N64 N64 2 2 4 4

64 N64 1 4 4 8

0.5 2 2 0.5 0.5 4

0.5/9.5 1/19 b0.5/9.5 b0.5/9.5 b0.5/9.5 b0.5/9.5

N4 N4 1 0.25 0.5 N4

6.5 6.5 22 40 37

Positive Positive Positive Negative Negative Positive

4 N64

2 16

N4 N4

12 18

Positive Positive

N64

4

2

2

N64

16

MIC (μg/mL) IDRL-8748 2 N16 IDRL-8890 0.125 N16 IDRL-8937 0.125 0.25 IDRL-8944 b0.125 b0.25 IDRL-8963 0.5 0.25 IDRL-9048 2 1 Two small colony variants from 1 subject IDRL-8826b 0.125 0.5 IDRL-8873b N16 N16 Small and normal colony pairs IDRL-8934 16 16 IDRL-8933c 0.125 0.25 IDRL-8866 0.25 0.5 c IDRL-8864 0.125 0.5 IDRL-8850 0.25 1 IDRL-8849c 0.25 1

0.125 0.5

0.5/9.5 0.5/9.5 N4/76 b0.5/9.5 0.5/9.5 0.5/9.5 2/38 1/19

2 2 2 2 0.5 0.5

N4 N4 N4 2 0.25 0.5

6.5

Positive

6.5

Positive

35

Positive

a Susceptibility testing by agar dilution for all antimicrobials tested except trimethoprim–sulfamethoxazole and cefoxitin. Trimethoprim–sulfamethoxazole susceptibility testing by Etest. b Small colony variants isolated from the same joint of a patient. 8826 grew as tiny clear colonies and 8873 as tiny white colonies; they had indistinguishable PFGE patterns. c Normal colony morphology.

3.4. Biofilm growth The biofilm indices of the positive and negative control at 48 h were 9.2 and 1.1, respectively. Of the 11 S. epidermidis SCV isolates tested, 2 (IDRL-8866 and IDRL-8890) were proficient biofilm formers showing biofilm indices higher than those of the positive control (12.9 and 11.7, respectively) at 48 h. Seven were poor biofilm formers with biofilm indices comparable to those of the negative control (≤1.2). Two (IDRL-8850 and IDRL-9048) were intermediate biofilm formers with 48 h biofilm indices of 6 and 5, respectively (Fig. 1, Table 2). Of the 3 SCVs that had co-isolated, clonally related, normal morphology isolates, 2 appeared to form more biofilm than their normal counterparts with 48 h biofilm indices of 12.9 versus 5.7 and 6 versus 3 for SCVs versus normal colony morphology isolates, respectively. However, there was no statistically significant difference in the total amount of biofilm formed over 72 h (P value for differences in biofilm indices between SCV and normal morphology of 0.12 [95% confidence interval (CI), −1.4 to 7.8] for 1 pair and 0.15 [95% CI, −0.82 to 3.65] for the other pair). 3.5. Transmission electron microscopy There were no differences noted between the S. epidermidis SCVs tested and their normal morphology counterparts with regard to cell

wall thickness, presence of ghost cells, and heterogeneity of cell shape and size. The S. aureus SCV used for comparison demonstrated several ghost cells, as well as heterogenous cell sizes, aberrant shapes, and thick cell walls. S. aureus ATCC 29213 did not show any ghost cells, and cells were of uniform shape and size.

4. Discussion Persistent and difficult-to-treat infections due to SCVs of S. aureus have been attributed (among other factors) to antimicrobial resistance which has been linked to auxotrophy for certain nutrients (typically hemin, menadione, or thymidine) and to enhanced formation of biofilm. We sought to determine whether SCVs of non-aureus staphylococci isolated from PJI demonstrated similar characteristics. Five of 11 S. epidermidis isolates demonstrated reduced susceptibility to at least 1 aminoglycoside. Of the 3 SCVs with co-isolated, clonally related normal morphology isolates, 1 demonstrated less aminoglycoside susceptibility than its normal counterpart, while 2 had similar aminoglycoside susceptibility to their co-isolated normal morphology isolates. The susceptibility to aminoglycosides and to TMP-SMX among the S. epidermidis isolates is in contrast to what has been reported for S. aureus SCVs and may relate to the finding that none of the S. epidermidis isolates was

Table 2 Antimicrobial susceptibility, auxotrophy, effect of serial passage, and biofilm-forming capacity of S. epidermidis SCV. Isolate number

Resistance to 1 or more aminoglycosides

Trimethoprim– sulfamethoxazole resistance

Auxotrophy to CO2, menadione, thymidine, or hemin

Stability with serial passagea

Fluctuating phenotype on serial passage

Biofilm phenotypeb

IDRL-8748 IDRL-8866 IDRL-8826 IDRL-8873 IDRL-8850 IDRL-8890 IDRL-8934 IDRL-8937 IDRL-8944 IDRL-8963 IDRL-9048

Yes No No Yes Yes Yes Yes No No No No

No No No No No No Yes No No No No

None None CO2 CO2 None None CO2 None None None None

Unstable Unstable Very unstable Very unstable Stable Stable Very unstable Unstable Very unstable Stable Very unstable

No Yes Yes No No No Yes No No No No

Poor Proficient Poor Poor Intermediate Proficient Poor Poor Poor Poor Intermediate

a Effect of passage: stable (remain SCV for ≥8 passages); unstable (revert to normal-sized colonies after 3–6 passages); very unstable (revert to normal-sized colonies after 1–2 passages). b Biofilm phenotype: poor—biofilm index (mean biofilm OD492/mean planktonic OD600) ≤2; intermediate—biofilm index 3–7; proficient—biofilm index ≥8.

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14

Biofilm index

12 10 8 6 4 2 0 6

12

24 Time (in hours)

48

72

ATCC 35984 blank IDRL-8944 IDRL-8937 IDRL-8963 IDRL-8748 IDRL-8890 IDRL-8850 IDRL-8866 IDRL-8873 IDRL-8826 IDRL-8934 IDRL-9048

Fig. 1. Biofilm growth of Staphylococcus epidermidis small colony variants compared to RP62A (ATCC 35984) and to uninoculated wells (blank).

auxotrophic for hemin or menadione (a phenotype typically associated with aminoglycoside resistance in S. aureus) or for thymidine (a phenotype typically associated with resistance to folate antagonists in S. aureus). This suggests that aminoglycoside resistance may not be a uniform property of staphylococcal SCVs but may be limited to SCVs of certain species and/or to SCVs that are menadione or hemin auxotrophic. More work is needed to identify alternative mechanisms behind the SCV phenotype in S. epidermidis. Of the 11 SCVs tested, 9 were positive for mecA of which only 8 were phenotypically resistant to oxacillin. This is consistent with what was previously reported by Pourmand et al. (2011) where only 61% of their mecA-positive S. epidermidis isolates (not SCVs) were phenotypically resistant to oxacillin. It is possible that, while mecA is present in these isolates, it is not expressed in some isolates, perhaps as a result of repressor mechanisms such as have been described for S. aureus (Ender et al., 2008; Hososaka et al., 2007). The enhanced biofilm-forming capacity (compared to non-SCV phenotypes) previously described for laboratory-derived menadione auxotrophs of S. aureus (Singh et al., 2009, 2010) was not observed among most of the clinical S. epidermidis SCVs tested, at least using the assay studied. With regard to the SCV-normal colony morphology pairs, while the biofilm index at 48 h appeared to be greater for the SCVs than for the normal colony morphology isolates, when the total amount of biofilm formed over 72 h (corrected for growth rate) was taken into account, there was no difference between the SCVs and the normal morphology isolates. Stepanović et al. (2007) proposed a classification scheme dividing isolates into categories of none, weak, moderate, and strong biofilm formers based on how their biofilm optical density compared with that of a negative control. In this scheme, a cutoff optical density was obtained by calculating a value 3 SD above the mean for the negative control. Classification was then based on where the biofilm OD for the test isolate fell relative to the cutoff optical density. With the Stepanović classification, our tested isolates would all have been classified as moderate or strong biofilm formers (data not shown). However, this classification (at least in the case of SCV) overestimates growth in the biofilm versus planktonic state. This is evidenced by the low biofilm index of these isolates, suggesting more prolific growth in the planktonic than in the biofilm state. This is likely because the Stepanović scheme basically compares growth of biofilm in inoculated wells with that in uninoculated wells (in which there should be no growth) as the basis of the classification. We therefore believe that use of the biofilm index, after Kristich et al. (2004) (as used herein), is a better reflection of biofilm versus planktonic growth. Our findings suggest that, while S. epidermidis causing PJI are able to exist as SCVs, similar in appearance to S. aureus SCVs, they do not necessarily share the auxotrophic characteristics, reduced aminogly-

coside susceptibility, or enhanced biofilm formation that has been described for SCVs of S. aureus. Thus, the SCV phenotype in itself may not predict these characteristics in non-aureus staphylococci. We also found that, as has been previously described for normal morphology isolates of S. epidermidis, phenotypic oxacillin susceptibility testing does not consistently predict the presence of mecA as some isolates will test susceptible to oxacillin and other β-lactams in the presence of mecA. Transmission electron microscopy did not show the ultrastructural characteristics noted by prior investigators in the SCV of enterococci. More research is needed to determine the impact of infection with SCVs of non-aureus staphylococci on patient outcome. Because of the differential antimicrobial susceptibility noted between some SCV and co-isolated normal morphology isolates, and between different morphology SCVs isolated from the same site, clinical microbiology laboratories should perform antimicrobial susceptibility testing on all colony morphologies present.

Acknowledgments The authors would like to thank the staff of Mayo Clinic Bacteriology and Infectious Disease Research Laboratories, particularly Dr. Adnan A. Alatoom, Scott A. Cunningham, Peggy C. Kohner, and Suzannah M. Schmidt, for their assistance with this study.

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