L E T T E R TO TH E ED I TOR

Vancomycin modulated autolysis in Staphylococcus aureus:does it vary with the susceptibility and planktonic or biofilm

phenotype?

DOI: 10.1111/j.1574-695X.2012.00951.x

Final version published online 2 April 2012.

We read the article by Hsu et al. (2011) with great interest.

The authors have convincingly demonstrated that bacterial

autolysis, and extracellular DNA (e-DNA) thereby released,

contributes to the enhanced biofilm-formation by vanco-

mycin nonsusceptible Staphylococcus aureus (VNSSA) upon

exposure to sublethal vancomycin concentration.

Many previous publications have also reported an

increased biofilm-formation by staphylococci upon expo-

sure to vancomycin (Dunne, 1990; Cargill & Upton,

2009; Kaplan et al., 2011; Mirani & Jamil, 2011). The

majority of these articles have focused on vancomycin-

susceptible strains, and studies specifically targeting

VNSSA biofilms (Mirani & Jamil, 2011) are rare. Also,

most studies have linked the antibiotic-enhanced Staphy-

lococcus biofilm-formation (especially Staphylococcus epi-

dermidis) to an increased production of polysaccharide

intercellular adhesin (PIA), the principal component of

biofilm matrix, perhaps via down-regulation of PIA

repressor TcaR (Rachid et al., 2000; Chang et al., 2011).

Interestingly, Hsu et al. (2011) demonstrate a role of

e-DNA for this mechanism in VNSSA. Although the pro-

duction of PIA was increased upon exposure to sublethal

vancomycin in one strain (SJC1200), e-DNA played a

more important role, with DNase significantly reducing

vancomycin-enhanced biofilm-formation to levels similar

to untreated cultures in both the strains tested (SJC1200

and Mu50) and xenogenic DNA promoting biofilm devel-

opment. These findings add considerably to the currently

available literature on S. aureus biofilms and perhaps

correlate with a greater role of e-DNA, rather than PIA,

in S. aureus biofilm development (Izano et al., 2008).

Another study has also implicated both PIA and e-DNA

for antibiotic-augmented biofilm-formation, depending

upon strain or drug tested, albeit in vancomycin-suscepti-

ble S. epidermidis (VSSE; Kaplan et al., 2011).

The authors report an increased bacterial autolysis in

VNSSA biofilms cultured in media containing sublethal

vancomycin; autolysis in planktonic cells was unaffected.

However, cid/lrg, the principal mechanism governing

autolysis in S. aureus, was found to be up-regulated both

in planktonic and biofilm cells grown in the presence of

vancomycin compared with unexposed controls. It is

intriguing to note that planktonic cells over-express cid/

lrg in the presence of vancomycin, but overall, the autoly-

sis remains unchanged. Also, many previous publications

have reported that VNSSA planktonic cells often inhibit

autolysis to subvert the antibiotic action (Sieradzki &

Tomasz, 1997, 2003). Thus, these discrete findings suggest

that VNSSA planktonic cells and biofilms perhaps exhibit

contrasting phenotypes in response to vancomycin.

Whilst autolysis is inhibited (or unaffected) in VNSSA

planktonic phase, it is considerably increased in biofilms.

Our studies on a vancomycin-susceptible S. aureus

(VSSA) strain, ATCC 29213, have further revealed an

additional facet of S. aureus biofilm response to vanco-

mycin. Treatment of 48-h-old S. aureus ATCC 29213 bio-

films [grown on black polycarbonate membranes (pore

size, 0.4 lm; diameter, 13 mm) placed on tryptic soy agar

plates] with 32 lg vancomycin mL�1 (~ MIC multiple

corresponding to clinically-achievable concentration) for

24 h resulted in reduced bacterial clumping relative to

untreated controls, as observed by scanning electron

microscopy (SEM; Singh et al., 2010a). In contrast,

VNSSA biofilms show an increased bacterial clumping in

response to vancomycin, as described by Hsu et al.

(2011) (SEM results). Also, further characterization of

vancomycin-treated VSSA ATCC 29213 biofilms by trans-

mission electron microscopy and autolysis quantification

reveal an inhibition of autolytic processes in biofilms after

vancomycin exposure, compared with untreated controls

(R. Singh and P. Ray, unpublished data). Whilst the total

number of bacteria mm�2 and bacterial cell wall thickness

in vancomycin-treated biofilms were similar to untreated

controls, bacterial cell diameter was significantly larger

(compared with untreated, P < 0.001), and an incomplete

cell division septum was observed in the treated biofilms,

especially in middle layers and near air interface (Fig. 1),

wherein the antibiotic penetration is expected to be less

(Singh et al., 2010b). Septum formation and cell division

are often associated with autolytic processes in staphylo-

cocci and their suppression results in increased cell wall

thickness and/or incomplete septation, inhibition of cell

separation and increased cell size, thereby leading to a

transient induction of vancomycin tolerance, following

FEMS Immunol Med Microbiol 65 (2012) 1–4 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

IMM

UN

OLO

GY

& M

EDIC

AL

MIC

ROBI

OLO

GY

exposure to subinhibitory doses, even in susceptible

strains (Sieradzki & Tomasz, 2006). Hence, these observa-

tions suggest an inhibition of autolysis in vancomycin-

treated S. aureus ATCC 29213 biofilms, possibly as a

mechanism of vancomycin tolerance in VSSA biofilms.

Also, these results were observed specifically with vancomycin,

Fig. 1. (a) Diagram indicating the model used for biofilm-formation. The biofilms were grown on black, polycarbonate membranes (diameter,

13 mm; pore size, 0.4 lm) placed on tryptic soy agar plates for 48 h at 37 °C. In this model, the antibiotic penetrates from the bottom of the

medium [bottom of the biofilm (membrane interface) towards the top (air interface)]. (b–e) Representative transmission electron micrographs (b),

total number of cells and dividing cells mm�2 (c), cell wall thickness (d, Mean ± SD) and cell diameter (e, Mean ± SD) near the air interface,

middle and the membrane interface of untreated (UNT) and vancomycin-treated (32 lg mL�1, 24 h; VAN-32) biofilms of Staphylococcus aureus

ATCC 29213 as analysed by transmission electron microscopy. Three locations within each colony biofilm were tested: near the air interface, in

the middle and near the membrane (memb.) interface. Whilst in the membrane interface, a few lysed cells were observed, the mean cell

diameter was significantly greater (P < 0.001) and incomplete septation was observed near the middle and air interface of the biofilms. Cell wall

thickness and cell diameter was determined at 10 cells selected randomly at each location. The experiment was repeated thrice to confirm the

observations. Magnification, 10 0009; Scale bar, 1 lm.

ª 2012 Federation of European Microbiological Societies FEMS Immunol Med Microbiol 65 (2012) 1–4Published by Blackwell Publishing Ltd. All rights reserved

2 Comment on Hsu et al., (2011)

and exposure of biofilms to amoxicillin, oxacillin, cefo-

taxime, amikacin and ciprofloxacin at clinically-achievable

levels did not cause increase in cell size or septum

inhibition (data not shown). However, Hsu et al. (2011)

demonstrate a significant increase in biofilm-formation

by VNSSA in response to some other antibiotics as well

(ampicillin, oxacillin and trimethoprim/sulfamethoxazole).

Another study has reported an augmentation in S. aureus

biofilm-formation by subinhibitory cefalotin, although the

mechanism involved was not characterized (Subrt et al.,

2011). Our findings on vancomycin were further confirmed

by phenotypic quantification of autolysis. Pretreatment of

S. aureus ATCC 29213 biofilms with 32 lg vancomy-

cin mL�1 for 24 h, and then suspension in autolysis buffer

[50 mM Glycine-Triton-X 100 (0.01%) buffer, pH 8.0;

24 h] caused a substantial reduction in per cent autolysis

(17.50%) compared with untreated biofilms (31.40%).

However, autolysis rate in planktonic cultures was

unaffected by pre-exposure to vancomycin (% autolysis:

treated, 21.25; untreated, 24.50).

Staphylococcal biofilm response to vancomycin is fur-

ther complicated by the data on S. epidermidis. In contrast

to VNSSA and VSSA, 48-h-old biofilms of a VSSE (ATCC

35984) exhibit spatial distribution in vancomycin-induced

damage, with the centre of the biofilms containing dam-

aged cells with sparse distribution, smaller size and irregu-

lar shape and the periphery remaining unaffected (Singh

et al., 2010a).

In conclusion, the presently available data suggest a

marked difference in the phenotype of VSSA and VNSSA

upon exposure to vancomycin, both in planktonic and bio-

film state. In VNSSA, autolysis is suppressed (or unaf-

fected) in planktonic cells, but enhanced in biofilms upon

exposure to vancomycin. On the contrary, autolysis is

inhibited in VSSA biofilms as a response to vancomycin: a

phenotype resembling the planktonic VNSSA, but remains

unaffected by vancomycin pre-exposure in planktonic

cultures. These results raise an important clinical concern

for the use of vancomycin against several S. aureus biofilm-

associated infections. However, the differences in experi-

mental set-up employed, antibiotic concentration chosen,

and strain backgrounds used in various studies may have

also contributed to the differences in the results obtained.

Further in vitro and in vivo comparative studies of VSSA

and VNSSA under same experimental conditions need to

be done to clarify the precise influence of vancomycin on

S. aureus biofilm-formation.

Acknowledgement

This work was supported by funding from Council of

Scientific and Industrial Research (CSIR), Govt. of India,

in the form of research fellowship for the first author.

References

Cargill JS & Upton M (2009) Low concentrations of

vancomycin stimulate biofilm formation in some clinical

isolates of Staphylococcus epidermidis. J Clin Pathol 62:

1112–1116.Chang YM, Jeng WY, Ko TP, Yeh YJ, Chen CK & Wang AH

(2011) Structural study of TcaR and its complexes with

multiple antibiotics from Staphylococcus epidermidis. P Natl

Acad Sci USA 107: 8617–8622.Dunne WM Jr (1990) Effects of subinhibitory concentrations

of vancomycin or cefamandole on biofilm production by

coagulase-negative staphylococci. Antimicrob Agents

Chemother 34: 390–393.Hsu CY, Lin MH, Chen CC, Chien SC, Cheng YH, Su IN &

Shu JC (2011) Vancomycin promotes the bacterial autolysis,

release of extracellular DNA, and biofilm formation in

vancomycin-non-susceptible Staphylococcus aureus. FEMS

Immunol Med Microbiol 63: 236–247.Izano EA, Amarante MA, Kher WB & Kaplan JB (2008)

Differential roles of poly-N-acetylglucosamine surface

polysaccharide and extracellular DNA in Staphylococcus

aureus and Staphylococcus epidermidis biofilms. Appl Environ

Microbiol 74: 470–476.Kaplan JB, Jabbouri S & Sadovskaya I (2011) Extracellular

DNA-dependent biofilm formation by Staphylococcus

epidermidis RP62A in response to subminimal inhibitory

concentrations of antibiotics. Res Microbiol 162: 535–541.Mirani ZA & Jamil N (2011) Effect of sub-lethal doses of

vancomycin and oxacillin on biofilm formation by

vancomycin intermediate resistant Staphylococcus aureus.

J Basic Microbiol 51: 191–195.Rachid S, Ohlsen K, Witte W, Hacker J & Ziebuhr W (2000)

Effect of subinhibitory antibiotic concentrations on

polysaccharide intercellular adhesin expression in biofilm-

forming Staphylococcus epidermidis. Antimicrob Agents

Chemother 44: 3357–3363.Sieradzki K & Tomasz A (1997) Inhibition of cell wall

turnover and autolysis by vancomycin in a highly

vancomycin-resistant mutant of Staphylococcus aureus.

J Bacteriol 179: 2557–2566.Sieradzki K & Tomasz A (2003) Alterations of cell wall

structure and metabolism accompany reduced

susceptibility to vancomycin in an isogenic series of

clinical isolates of Staphylococcus aureus. J Bacteriol 185:

7103–7110.Sieradzki K & Tomasz A (2006) Inhibition of the autolytic

system by vancomycin causes mimicry of vancomycin-

intermediate Staphylococcus aureus-type resistance, cell

concentration dependence of the MIC, and antibiotic

tolerance in vancomycin-susceptible S. aureus. Antimicrob

Agents Chemother 50: 527–533.Singh R, Ray P, Das A & Sharma M (2010a) MRT letter:

Spatial distribution of vancomycin-induced damage in

Staphylococcus epidermidis biofilm: an electron microscopic

study. Microsc Res Tech 73: 662–664.

FEMS Immunol Med Microbiol 65 (2012) 1–4 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Letter to the Editor 3

Singh R, Ray P, Das A & Sharma M (2010b) Penetration

of antibiotics through Staphylococcus aureus and

Staphylococcus epidermidis biofilms. J Antimicrob Chemother

65: 1955–1958.Subrt N, Mesak LR & Davies J (2011) Modulation of

virulence gene expression by cell wall active antibiotics in

Staphylococcus aureus. J Antimicrob Chemother 66:

979–984.

Rachna Singh and Pallab RayDepartment of Medical Microbiology

Postgraduate Institute of Medical Education and Research(PGIMER) Chandigarh, India

E-mail: rachnasingh.25@gmail.com

ª 2012 Federation of European Microbiological Societies FEMS Immunol Med Microbiol 65 (2012) 1–4Published by Blackwell Publishing Ltd. All rights reserved

4 Comment on Hsu et al., (2011)