Staphylococcus epidermidis is a cause of orthopedic device-related infection, and to treat such infection, biofilms should be controlled. Polysaccharide intercellular adhesin (PIA) is associated with the biofilm-forming ability of staphylococcal strains. PIA in biofilm-positive staphylococcal strains can be detected by the Congo red agar (CRA) method. In this study, we used the CRA method to examine the effects of subminimal inhibitory concentrations (sub-MICs) of 11 antibacterial agents on PIA production by S. epidermidis. We found that the PIA-negative variants were selected only by sub- MICs of gentamicin (GM). This PIA-negative phenotype was maintained over several generations in the absence of GM. Such selection occurred in six of eight clinical isolates, as well as in the biofilm-positive control strain. No such selection occurred with aminoglycoside antibiotics except for GM. Most of the PIA-negative variants that were selected by GM showed a markedly lower biofilm-forming ability on stainless steel washers than their untreated parent strains. In conclusion, variants with lower biofilmforming ability may be selected by a sub-MIC of GM. Investigation of the reason why variants with reduced biofilm-forming ability can be selected in the presence of sub-MICs of GM may contribute to strategies against biofilm-related infections

Adachi Shinji

Kajiyama Shiro

Sakimura Toshiyuki

Shindo Hiroyuki

Tamai Takashi

Tsurumoto Toshiyuki

Nagasaki University's Academic Output SITE: NAOSITE

304*Corresponding author: Mailing address: Department ofOrthopedic Surgery, Graduate School of BiomedicalScience, Nagasaki University, 1-7-1, Sakamoto, Nagasaki852-8501, Japan. E-mail: t.tamai＠ind.bbiq.jpJpn. J. Infect. Dis., 64, 304-308, 2011Original ArticleBiofilm Deficiency in Polysaccharide Intercellular Adhesin-NegativeVariants of Staphylococcus epidermidis Selected bySubminimal Inhibitory Concentrations of GentamicinTakashi Tamai*, Toshiyuki Tsurumoto1, Shiro Kajiyama,Shinji Adachi, Toshiyuki Sakimura, and Hiroyuki ShindoDepartment of Orthopedic Surgery and 1Department of Gross Anatomy, Graduate School ofBiomedical Science, Nagasaki University, Nagasaki 852-8501, Japan(Received August 9, 2010. Accepted May 11, 2011)SUMMARY: Staphylococcus epidermidis is a cause of orthopedic device-related infection, and to treatsuch infection, biofilms should be controlled. Polysaccharide intercellular adhesin (PIA) is associatedwith the biofilm-forming ability of staphylococcal strains. PIA in biofilm-positive staphylococcalstrains can be detected by the Congo red agar (CRA) method. In this study, we used the CRA method toexamine the effects of subminimal inhibitory concentrations (sub-MICs) of 11 antibacterial agents onPIA production by S. epidermidis. We found that the PIA-negative variants were selected only by sub-MICs of gentamicin (GM). This PIA-negative phenotype was maintained over several generations in theabsence of GM. Such selection occurred in six of eight clinical isolates, as well as in the biofilm-positivecontrol strain. No such selection occurred with aminoglycoside antibiotics except for GM. Most of thePIA-negative variants that were selected by GM showed a markedly lower biofilm-forming ability onstainless steel washers than their untreated parent strains. In conclusion, variants with lower biofilm-forming ability may be selected by a sub-MIC of GM. Investigation of the reason why variants withreduced biofilm-forming ability can be selected in the presence of sub-MICs of GM may contribute tostrategies against biofilm-related infections.INTRODUCTIONOrthopedists want to ensure the eradication ofpostoperative infections. Recently, biofilms producedby bacteria have attracted attention as a cause of chron-ic and intractable device-related infections (1). It hasbeen suggested that the bacteria in biofilms are 100- to1,000-fold more resistant to antibacterial agents thanplanktonic bacteria (2). It has also been suggested thatthe biofilm-forming ability of some staphylococcalstrains is dependent in part on polysaccharide intercellu-lar adhesin (PIA). Various reports have been publishedon the effects of subminimal inhibitory concentrations(sub-MICs) of antibacterial agents on biofilm formationand PIA production; however, the results are still con-troversial.Here, we investigated whether sub-MICs of 11 anti-bacterial agents affect PIA production, and whether al-tered PIA production reflects biofilm-forming abilityusing a biofilm-positive control strain and eight clinicalisolates.MATERIALS AND METHODSBacterial strains and chemicals: The eight clinical iso-lates of Staphylococcus epidermidis used in this studywere obtained from infected sites in patients with boneand joint infections at Nagasaki University Hospital. S.epidermidis American Type Culture Collection (ATCC)35984 (RP62A), which is a well-known biofilm producer(3), was used as a positive control for biofilm forma-tion. These strains were stored in beads (MicroBankTM;Pro-Lab Diagnostics, Richmond Hill, Canada) at－809C. Congo red (1z Congo red solution) was pur-chased from Muto Pure Chemicals (Tokyo, Japan).Mueller-Hinton broth (MHB) and trypticase soy broth(TSB) were obtained from Becton-Dickinson and Com-pany (Sparks, Md., USA). Agar was purchased fromIna Food Industry (Nagano, Japan). D-(＋)-glucose wasobtained from Wako Pure Chemical Industries (Osaka,Japan). Crystal violet (CV, 1z Pyoktanin Blue Solu-tion) was purchased from Kanto Chemical (Tokyo,Japan). Phosphate-buffered saline (PBS) and bloodagar were purchased from Gibco (Gaithberg, Md.,USA) and Eiken (Tochigi, Japan), respectively.Antibacterial agents used in this study and determina-tion of minimal inhibitory concentrations (MICs) ofgentamicin (GM): The antibacterial agent-containingdiscs (BBL Sensi-Disc) used in this study were purchasedfrom Becton-Dickinson Company, and the amount ofeach agent per disc was as follows: GM, 120 mg; amika-cin (AMK), 30 mg; arbekacin (ABK), 30 mg; vancomycin(VCM), 30 mg; tetracycline (TC), 30 mg; chloram-phenicol (CP), 30 mg; cefazolin (CEZ) 30 mg; fosfomy-cin (FOM) 50 mg; minocycline (MINO) 30 mg; rifampin(RFP), 5 mg; and clarithromycin (CAM), 15 mg.MICs for suspended bacteria were measured by themicrodilution method (4).305Fig. 1. The effects of subminimal inhibitory concentrations (sub-MICs) of gentamicin (GM) on polysaccharide in-tercellular adhesin (PIA) production were assessed using Congo red agar (CRA) method. (A) ATCC 35984, (B)strain no. 1, (C) strain no. 2, (D) strain no. 3, (E) strain no. 4, (F) strain no. 5, (G) strain no. 6, (H) strain no. 7, (I)strain no. 8. PIA-negative variants grow as red colony on CRA. PIA-negative variants appeared around inhibitoryzones for all strains, with two exceptions (B and C).Assessment of PIA production using the Congo redagar (CRA) method: PIA production was assessed usingthe CRA method as previously described (5). Briefly,colonies on blood agar were inoculated into 10 mL ofTSB and incubated at 379C for approximately 3 h(OD600 ＝ 0.2). This culture was vortexed and then dilut-ed 10-fold with PBS. Approximately 0.1 mL of thediluted suspension was dropped onto CRA plates(0.08z Congo red, 2.1z MHB, 0.5z glucose, and1.7z agar), the plates were briefly air-dried, and thenthe antibacterial discs were put in place. The plates wereincubated at 379C for 48 h and then incubated at roomtemperature for 24 h. PIA production was determinedby macroscopic observation since PIA-negative variantsform red colonies, whereas PIA-positive variants formblack colonies (5). PIA-negative variants were used inthe subsequent experiments.Assessment of biofilm formation: The biofilm-form-ing ability of the PIA-negative variants was comparedwith that of their cognate parent strains using a previ-ously described method (6). Briefly, colonies were in-oculated in antibacterial agent-free TSB and incubatedat 379C for approximately 3 h (OD600 ＝ 0.2). Sterilizedstainless steel washers (UW–0303–05; 6.0 mm diameterand 0.5 mm thickness, SUS304 quality containing 18zchrome and 8z nickel; Wilco, Tokyo, Japan) were im-mersed in these cultures for 10 min to allow bacteria toadhere. Subsequently, the bacteria adhering to thewashers were incubated in fresh TSB at 379C for 24 hwith the adhered surface down in order to facilitatebiofilm formation.The washer to which the biofilms adhered was washedgently with PBS to remove the planktonic bacteria, andthen dried with a drier for approximately 5 min to fixthe bacteria to the washer. The biofilm adhering to thewasher was stained with CV for 2 min, and then washedtwice with PBS to remove the excess stain.Eight areas (660 mm × 480 mm) of each washer werearbitrarily selected and full-color pictures were obtainedusing a digital optical microscope (VHX-100; Keyence,Osaka, Japan). The biofilm coverage ratio (BCR) wascalculated as the ratio of the CV-stained area to totalarea of the washer. The color photograph was then con-verted to a grayscale TIFF image using Photoshop Ele-ments 6 (Adobe Systems, San Jose, Calif., USA), andthe BCR was measured with Scion Imaging software(Scion, Frederick, Md., USA). The data are presentedas percentages and the mean ± standard deviation (SD)of four replicates. The data were analyzed usingStudent's t test.Assessment of revertants from serial passage of PIA-negative variants: PIA-negative variants underwent seri-306Fig. 2. Assessment of biofilm-forming ability of PIA-negative variants compared with that of the cognate parentstrains using crystal violet (CV) staining of biofilms formed on stainless steel washers. The macroscopic images ofthe CV-stained biofilms formed on stainless steel washers showed that the PIA-negative variant (B) impaired thebiofilm-forming ability compared with the parent strain of ATCC35984 (A). The biofilm coverage ratios (BCRs)were compared between the PIA-negative variants and the cognate parent strains derived from the PIA-positivecontrol strain (ATCC35984) and clinical strains in which sub-MICs of GM selected PIA-negative variants (C).*P º 0.05 compared with parent strains. **P º 0.01 compared with parent strains.al passage every 24 h in antibacterial-free TSB untilPIA-positive prototypes (revertants) appeared (for amaximum of 2 weeks). The CRA method describedabove was also used to determine reversion. Revertantswere identified when more than approximately 10z ofthe colonies that appeared were PIA-positive.Sodium chloride (NaCl) (maximum, 3z) has beenreported to promote reversion of some variants (7). Toassess the effect of NaCl, serial passages were per-formed using antibacterial-free TSB with 0.5z NaCluntil reversion occurred (for a maximum of 2 days).RESULTSPIA-negative variants appear at sub-MICs of GM:The MICs of GM against clinical strain No. 1, 2, 3, 4, 5,6, 7, and 8 and strain ATCC35984 were Ã0.5, Ã0.5,Æ32, Æ32, Æ32, Æ32, 16, Æ32, and 8 mg/mL, respec-tively. PIA-negative variants appeared around the GMinhibitory zone of neally all strains, with two exceptions(Fig. 1). In contrast, PIA-negative variants did not ap-pear around the inhibitory zone of the other antibacteri-al agents used in this study (data not shown).This PIA-negative phenotype was maintained in theabsence of GM over several generations.PIA-negative variants have impaired biofilm-formingability compared with their cognate parent strains: Themacroscopic images of CV-stained biofilms formed onstainless steel washers showed that the PIA-negativevariant of ATCC35984 (Fig. 2B) had impaired biofilm-forming ability compared with the parent strain (Fig.2A). The PIA-negative variants of the clinical isolatesalso had macroscopically impaired biofilm-formingability compared with the parent strains. The BCRs of307the PIA-negative variants, except for No. 5, were sig-nificantly lower than those of their cognate parentstrains, and the BCR of strain No. 5 also tended to belower than that of the parent strain (P ＝ 0.056) (Fig.2C).PIA-negative variants reverted to the PIA-positivephenotype after serial passages; however, reversion wasnot promoted by NaCl: PIA-negative variants revertedto the PIA-positive phenotype after four or more pas-sages. ATCC35984 reverted after 10 passages, No. 4, 5,6, 7, and 8 reverted after 12 or 13 passages, and No. 3reverted after four passages. NaCl did not promote thereversion of any strain tested in our study.DISCUSSIONAlthough Rachid et al. previously reported that sub-MICs of GM had no effect on the expression of the icagenes that encode enzymes involved in PIA production(8), we found that PIA-negative variants were selectedby sub-MICs of GM. It is unlikely that the PIA-negativevariants are produced due to the action of Congo red atthe concentration employed in the CRA method (9).Although the method used in our study is different fromthat used in their study, we confirmed that PIA-negativevariants were selected by GM in the absence Congo red(data not shown). Therefore, we could not clarify thediscrepancy between these studies; however, it was notdependent on the combination of GM and Congo redthat we used.The mechanism by which PIA-negative variants areselected by sub-MICs of GM is not fully understood;however, in a previous report by Ziebuhr et al., threepossibilities were suggested. They classified naturallyoccurring PIA-negative variants into three groups. Inthe first group, the PIA-negative variants revert to thePIA-positive phenotype after only two serial passages orwith the addition of NaCl, and expression of the icaoperon is inhibited (7). In the second group, 9 to 12 seri-al passages are required for PIA-negative to PIA-posi-tive reversion (7); this group includes phase variantsproven to be caused by rearrangement of the ica operon(10,11). In the third group, reversion never occurs, andthe ica operon is either completely or partially deleted(7). According to their classification, our variants mightbelong to the second group; however, gene arrangementwas not confirmed, and further investigation is re-quired.The reason why PIA-negative variants were not ob-tained from two of the clinical isolates is unclear.However, the sensitivities of these strains to GM werehigher (MICs Ã0.5 mg/mL) than those of other strains.Therefore, for the two clinical isolates from which noPIA variant was selected, the antibacterial effect of GMmay have been dominant to the PIA-negative variant-selecting effect.We also showed that most PIA-negative variants hadsignificantly impaired biofilm-forming ability. Accord-ingly, our results suggest that the impaired biofilm-forming ability of these variants may be due in part tothe inhibition of PIA production.Because inhibition of protein synthesis is the antibiot-ic mechanism of action for GM, we initially consideredthat inhibition of protein synthesis resulted in the selec-tion of PIA-negative variants of the first group. It hasalso been reported that a protein synthesis inhibitor,CP, inhibited biofilm synthesis (12). However, ourstudy showed that PIA-negative variants were notselected by CP or other aminoglycosides except GM.Therefore, the impaired biofilm-forming ability of ourPIA-negative variants might be independent of proteinsynthesis inhibition. In addition, biofilm production isdependent on not only PIA production due to ica geneexpression but also agr genes and other factors (13).Furthermore, ica-independent biofilm-forming strainshave recently been reported (14). These complicatedmechanisms might also contribute to the variation inbiofilm-forming ability among our PIA-negative vari-ants.There was no significant difference between the BCRsof the parent strain and the PIA-negative variant of No.5. We employed the BCR method because our laborato-ry is familiar with this method, and it is relatively relia-ble and reproducible (15). However, this method doesnot reflect the three-dimensional mass of biofilms, andthus it might have underestimated significant differ-ences in biofilm-forming ability between the parentstrain and the PIA-negative variant of isolate No. 5.In conclusion, we reported that PIA-negative variantsselected by sub-MICs of GM markedly impairedbiofilm-forming ability. Our finding is clinically intri-guing because biofilm formation frequently causes in-tractable infections. The mechanism underlying biofilmformation should be determined in order to provide ef-fective anti-biofilm strategies against intractable infec-tions.Acknowledgments We are grateful to Katsunori Yanagihara(Division of Laboratory Medicine, Nagasaki University Hospital,Nagasaki, Japan) for storing and supplying strains.Conflict of interest None to declare.REFERENCES1. Stewart, P.S. and Costerton, J.W. (2001): Antibiotic resistance ofbacteria in biofilms. Lancet, 358, 135–138.2. Gristina, A.G. and Costerton, J.W. (1985): Bacterial adherenceto biomaterials and tissue: the significance of its role in clinicalsepsis. J. Bone Joint Surg. Am., 67, 264–273.3. G äotz, F. (2002): Microreview Staphylococcus and bioflims. Mol.Microbiol., 43, 1367–1378.4. Clinical and Laboratory Standards Institute (2009): Methods fordilution antimicrobial susceptibility tests for bacteria that growaerobically; approved standard-8 ed. Document M7-A8. Clinicaland Laboratory Standards Institute, Wayne, Pa.5. Freeman, D.J., Falkiner, F.R. and Keane, C.T. (1989): Newmethod for detecting slime production by coagulase negativestaphylococci. J. Clin. Pathol., 42, 872–874.6. Adachi, K., Tsurumoto, T., Yonekura, A., et al. (2007): Newquantitative image analysis of staphylococcal biofilms on the sur-faces of nontranslucent metallic biomaterials. J. Orthop. Sci., 12,178–184.7. Ziebuhr, W., Loessner, I., Krimmer, V., et al. (2001): Methods todetect and analyze phenotypic variation in biofilm-formingstaphylococci. Methods Enzymol., 336, 195–205.8. Rachid, S., Ohlsen, K., Witte, W., et al. (2000): Effects of subin-hibitory antibiotic concentrations on polysaccharide intercellularadhesin expression in biofilm-forming Staphylococcus epidermi-dis. Antimicrob. Agents Chemother., 44, 3357–3363.9. Arciola, C.R., Campoccia, D., Gamberini, S., et al. (2002): De-tection of slime production by means of an optimised Congo redagar plate test based on a colourimetric scale in Staphylococcus308epidermidis clinical isolates genotyped for ica locus. Biomaterials,23, 4233–4239.10. Ziebuhr, W., Krimmer, V., Rachid, S., et al. (1999): A novelmechanism of phase variation of virulence in Staphylococcusepidermidis: evidence for control of the polysaccharide intercellu-lar adhesin synthesis by alternating insertion and excision of theinsertion sequence element IS256. Mol. Microbiol., 32, 345–356.11. Ziebuhr, W., Dietrich, K., Trautmann, M., et al. (2000): Chro-mosomal rearrangements affecting biofilm production and an-tibiotic resistance in a Staphylococcus epidermidis strain causingshunt-associated ventriculitis. Int. J. Med. Microbiol., 290,115–120.12. Dobinsky, S., Kiel, K., Rohde, H., et al. (2003): Glucose-relateddissociation between icaADBC transcription and biofilm expres-sion by Staphylococcus epidermidis: evidence for an additionalfactor required for polysaccharide intercellular adhesin synthesis.J. Bacteriol., 185, 2879–2886.13. Boles, R.B. and Horswill, A.R. (2008): agr-mediated dispersal ofStaphylococcus aureus biofilms. PLoS Pathog., 4, e1000052.14. O'Gara, J.P. (2007): ica and beyond: bioflm mechanisms andregulation in Staphylococcus epidermidis and Staphylococcusaureus. FEMS Microbiol. Lett., 270, 179–188.15. Kajiyama, S., Tsurumoto, T., Osaki, M., et al. (2009): Quantita-tive analysis of Staphylococcus epidermidis biofilm on the surfaceof biomaterial. J. Orthop. Sci., 14, 769–775.

Biofilm deficiency in polysaccharide intercellular adhesin-negative variants of staphylococcus epidermidis selected by subminimal inhibitory concentrations of gentamicin

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Biofilm deficiency in polysaccharide intercellular adhesin-negative variants of staphylococcus epidermidis selected by subminimal inhibitory concentrations of gentamicin

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