- Email: [email protected]
Slime production is essential for the adherence of Staphylococcus epidermidis in implant-related infections
Journal of Hospital Infection 77 (2011) 153e156
Available online at www.sciencedirect.com
Journal of Hospital Infection journal homepage: www.elsevierhealth.com/journals/jhin
Slime production is essential for the adherence of Staphylococcus epidermidis in implant-related infections N. Nayak a, G. Satpathy a, *, H.L. Nag b, P. Venkatesh c, S. Ramakrishnan d, T.C. Nag e, S. Prasad a a
Department of Ocular Microbiology, Dr Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India Department of Orthopaedic Surgery, All India Institute of Medical Sciences, New Delhi, India c Uvea & Retina Services, Dr Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India d Department of Cardiology, All India Institute of Medical Sciences, New Delhi, India e Department of Anatomy, All India Institute of Medical Sciences, New Delhi, India b
a r t i c l e i n f o
s u m m a r y
Article history: Received 20 August 2009 Accepted 10 September 2010 Available online 7 January 2011
A total of 32 Staphylococcus epidermidis isolates from indwelling device-related infections such as endophthalmitis following intraocular lens (IOL) implantation, intravenous catheter-related sepsis and orthopaedic implant infections, were studied for slime production and adherence to artificial surfaces. Of these, 21 (65.6%) isolates were slime positive by the Congo Red agar method and 24 (75%) were adherent to artificial surfaces by the quantitative slime test. The majority (19 out of 24; 79.1%) of the adherent bacteria were slime producers. Antibody to slime raised in rabbits was able to inhibit the adherence of all 24 bacteria designated as adherent by our quantitative test. It seems that slime is indispensable for the sessile mode of attachment, leading further to the development of biofilms on the indwelling devices. Ó 2010 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.
Keywords: Adherence Biofilms Device-related infections Implant-related infections Slime Staphylococcus epidermidis
Introduction
Methods
Slime is an exopolysaccharide liberated by Staphylococcus epidermidis.1 The clinical significance of slime from S. epidermidis in device-associated infections has been documented.2 Slime has also been reported as a virulence factor of S. epidermidis in bacterial keratitis.3,4 Clinical and laboratory evidence have supported the view that slime-producing S. epidermidis isolates from cases of keratitis are adherent to artificial surfaces.3,5 The molecular basis of slime production was established by amplification of the ‘ica’ locus using polymerase chain reaction assay in S. epidermidis isolates from central venous catheter-related sepsis.6 Despite all of the clinical, epidemiological, laboratory and molecular data, the exact role of slime as an adhesin has not been elucidated.2e6 The present study was undertaken to assess whether such slime exopolysaccharide produced by S. epidermidis in implant-associated infections has any direct adhesion potential, which might be involved in the process of attachment on to the indwelling implants.
Subjects and case definitions
* Corresponding author. Address: Dr Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi 110029, India. Fax: þ91 11 26588919. E-mail address: [email protected] (G. Satpathy).
Samples were collected from 73 patients with indwelling-implant related infections. They included 39 subjects of postoperative endophthalmitis following intraocular lens (IOL) implantation, 24 subjects with intravascular catheter-related sepsis, and 10 having infections due to implanted orthopaedic devices. Individuals with features of endophthalmitis such as increasing pain and redness, decreasing visual acuity, flare in the anterior chamber, corneal oedema, hypopyon, and poor glow within four to six weeks following IOL implantation were classified as cases of late-onset postoperative endophthalmitis (POE). All 39 subjects fulfilled the above criteria. The 24 patients with indwelling central venous catheters/ intravascular cannulas had clinical evidence of infection (catheterrelated sepsis) with fever 38 C, pulse rate of >90/min, respiration rate of >20/min and white blood cell count of >12 000/mm3 of blood. In addition, subjects with localised infection on the exit of the truncated tract were also considered as having catheter-related infections. Those who were having either localised inflammatory signs at the implant site or signs of sepsis, as defined above, owing to
0195-6701/$ e see front matter Ó 2010 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jhin.2010.09.023
154
N. Nayak et al. / Journal of Hospital Infection 77 (2011) 153e156
implantation of joint prostheses, nails, plates and bone cements were said to have orthopaedic implant-associated infections. Sample collection and processing Patients with POE About 0.1 mL of vitreous fluid was collected with the help of sterile tuberculin syringe and 26 gauge needle. The bevelled tip of the needle was closed with a sterile rubber bung and was transported to the laboratory immediately. The vitreous fluid was stained and cultured according to the standard procedures. Patients with intravascular implants The tips of intravascular catheters/cannulas and/or central venous catheters were collected aseptically, in sterile test tubes, and were immediately inoculated on to blood agar plates according to the rolling technique of Maki et al. and subsequently on to trypticase soy broth (TSB).7 The agar plates and TSB tubes were incubated at 37 C. For blood culture, the sample of blood was inoculated directly on to the broth. Patients with orthopaedic implants Aspirate was collected with sterile syringe and needle after properly disinfecting the surrounding skin. Pus or discharge was collected by rubbing the bed of the ulcer with a sterile cottontipped swab. If the material was insufficient, then the wound was squeezed and the exuded purulent material was collected. Intraoperative pus, if obtained, was directly inoculated on to TSB. Blood was also collected for culture if there was indication of sepsis. Additionally, the pus, aspirate, discharge were subjected to Gram staining. The material inoculated into TSB was incubated at 37 C. Growth from TSB was subcultured on to blood agar, MacConkey agar and chocolate agar plates, which were incubated at 37 C. Identification After overnight incubation, those colonies showing Grampositive cocci on smear examination were processed further. The organisms were identified and speciated according to the previously described method and were stored at e20 C as nutrient agar stab cultures until further testing.4 Slime test Test for slime production was carried out by the Congo Red agar (CRA) plate method described earlier.8
the mean OD of 10 blank cuvettes stained by the above-described procedure in that particular batch of tests.
Extraction of slime Crude slime was extracted by a procedure previously described.9 Briefly, 50 mL of mid-log phase of bacterial suspension were inoculated in 1 L of TSB and incubated for 24 h at 37 C, in humidified chambers. Extracellular material was removed from the cells by gentle shaking with glass beads in 0.15 M NaCl. The extracts were precipitated with a mixture of ethanol, sodium acetate and acetic acid in final concentrations of 80%, 0.26 M and 0.05 M respectively. The precipitate was dissolved in distilled water, the insoluble material was removed by centrifugation at 27 000 g for 30 min, and the supernatant was dialysed three times against 100 volumes of distilled water. The above procedure was repeated twice. The dialysates were centrifuged at 105 000 g for 3 h and the supernatants were freeze-dried and stored at e70 C. All of the above steps were performed at 4 C in the presence of 6-aminohexanoic acid, EDTA and phenylmethane sulphonylfluoride at final concentrations of 50, 10 and 20 mM respectively.
Antibodies to slime Rabbit antiserum to slime was prepared by immunisation of the animals by a protocol earlier adapted by Karamanos et al. with some modifications.10 Filter-sterilised antigen, i.e. crude slime, was emulsified with equal volumes of Freund’s complete adjuvant for the first injection subcutaneously and incomplete Freund’s adjuvant for the subsequent challenges one week later intravenously thrice weekly for three weeks. Animals were bled five days after final injection and antibody titres were measured by enzymelinked immunosorbent assay.
Adherence inhibition test Quartz cuvettes were coated with 1:20 dilution of the immune sera at 4 C overnight, by addition of 1 mL of diluted serum to each cuvette. Subsequent steps followed were the same as those described for the quantitative adherence test, the only exception being that bacterial suspension was added to antibody-coated cuvettes.5 The OD values recorded after such experiments (adherence inhibition test) were plotted for comparison with those recorded earlier (adherence test).
Results Quantitative slime test for adherence Adherence of each isolate to smooth surfaces was determined quantitatively by a method earlier standardised in our laboratory.5 Briefly, our procedure was as follows. Overnight cultures of bacteria in trypticase soy broth (TSB) were diluted 1 in 100 in fresh TSB, and 1 mL volume of each isolate was put into separate quartz cuvettes. After overnight incubation at 37 C, the cuvettes were washed four times with phosphate-buffered saline (PBS), fixed with Bouin’s fluid, and stained with crystal violet. Excess stain was removed by decanting the tubes and then rinsing them gently with tap water. The optical density (OD) of the stained adherent bacterial film was read with a spectrocolorimeter (Spectrocolorimeter 103, Systronics, Baroda, India) at 570 nm. Organisms were considered non-adherent if the OD was below the cut-off recorded for that batch of experiments. This cut-off was calculated as 3 SD above
Culture positivity in samples collected from patients with various implants Overall culture positivity was found in 43 (58.9%) samples, out of which S. epidermidis grew in 32 (28 as pure growth and four as mixed growth) (Table I). Other organisms recovered were: five isolates of diphtheroids from the vitreous fluid; two Pseudomonas aeruginosa, one Candida albicans and two viridans streptococci, all from intravascular catheters/cannulas; and a single isolate of Staphylococcus aureus from a pus sample in a patient with orthopaedic implant. All blood cultures were sterile. However, other organisms isolated as mixed growth along with S. epidermidis were Escherichia coli in two specimens (one from catheter and the other from pus), S. aureus in one and P. aeruginosa and E. coli in one specimen.
N. Nayak et al. / Journal of Hospital Infection 77 (2011) 153e156
155
Table I Samples collected and culture positivity among patients with various implants Implant type (no. of patients) and nature of the specimen Intraocular lens implant (39) Vitreous Cardiac implant (24) Catheter/cannula
Blood Orthopaedic implants (10) Pus/wound aspirate Blood Total a b
No. of samples
Culture positive
Culture positive for S. epidermidis alone
39
19
14
24
14
8
3a 10
Sterile 10
2b 73
Culture positive for other organisms as pure growth (no.)
Mixed growth (no.) with S. epidermidis
Diphtheroids (5)
Nil E. coli (1)
Sterile 6
P. aeruginosa (2) C. albicans (1) Viridans streptococci (2) Sterile S. aureus (1)
Sterile
Sterile
Sterile
Sterile E. coli (1) S. aureus (1) P. aeruginosa and E. coli (1) Sterile
43
28
11
4
Belong to the group of 24 cardiac patients. Belong to the group of 10 orthopaedic patients.
Phenotypic characteristics of S. epidermidis Phenotypic production of slime by all the strains was assessed by culture on CRA plates. Slime-producing strains exhibited black shiny colonies with a metallic tinge, whereas non-slime producers appeared pink (Figure 1). Of the 32 S. epidermidis isolates studied, 21 (65.6%) were slime positive (Table II). A total of 19 (90.5%) out of the 21 slime-producing organisms were adherent to artificial surfaces. By contrast, only 5 (45.4%) of the 11 non-slime producers were found to be adherent (P < 0.01). Adherence inhibition test Figure 2 shows the results of the adherence inhibition tests performed on all the 24 adherent bacteria, each plot representing an individual isolate. The middle curve denotes the cut-off OD values for the number of tests carried out at different time points.
On coating the cuvettes with slime antibody and thereafter performing the adherence test as per our protocol detailed above, there were significant diminutions in the individual OD values from those recorded without any antibody treatment (Figure 2; upper vs lower curve). This suggests that slime antibody successfully blocked the adherence of the organisms on to the artificial surfaces. Discussion During the last two decades, S. epidermidis has emerged as a major cause of nosocomial infection. This organism, which constitutes the main component of the normal skin and mucosal microflora, is particularly responsible for catheter and other medical device-related sepsis.4 Slime as a virulence factor for S. epidermidis in medical implant/device-related infections has been documented.2,11 In addition, the gene responsible for biofilm production, i.e. the ica operon, was identified in the majority of S. epidermidis strains from catheter-related sepsis, whereas none of the saprophytic strains possessed this intercellular adhesin gene.6 Despite all the above relevant documentations relating to the slime and its association with bacterial colonisation of the indwelling devices, the precise role of slime as an adhesin had not yet been established. The present study showed that 65.6% (21/32) of the strains were slime positive. By contrast, Arciola et al. reported 48.5% (33 out of 68) of their clinical isolates of S. epidermidis as slime positive.6 We also observed previously that about 43% of the corneal ulcer isolates of S. epidermidis were slime producers.3,4 The relatively high rate of slime positivity among our present isolates compared with those in our previous studies on keratitis, might suggest that S. epidermidis colonising the indwelling device may be more virulent than those colonising the corneal surface.3 Whereas Arciola et al. studied slime production on catheter isolates only; ours were not only from vascular devices, but also from others such as IOLs and orthopaedic implants.6 Thus it may be that in different microenvironments the organism behaves differently in expressing its
Table II Adherence properties of 32 S. epidermidis isolates
Figure 1. Congo Red agar test showing slime-positive bacteria with dark shiny colonies (upper left sector), slime negative bacteria showing pink-coloured colonies (upper right and lower sectors).
S. epidermidis isolates
Adherent
Slime positive Slime negative
19 5
2 6
21 11
Total
24 (75%)
8 (25%)
32
c2 ¼ 7.8, P < 0.01.
Non-adherent
Total
156
N. Nayak et al. / Journal of Hospital Infection 77 (2011) 153e156
adhesion leading eventually to the formation of biofilm, which is the rigid sessile form of the bacterial community.11,12
0.7 0.6
Conflict of interest statement None declared.
OD570
0.5 0.4 0.3
Funding sources Supported by a grant from Indian Council of Medical Research.
.
0.2 0.1
References
0 0
20 10 OD values of tests with 24 adherent S. epidermidis isolates
30
Figure 2. Adherence inhibition test result of all the 24 adherent organisms showing diminution of respective optical density (OD) values after antibody treatment (:); cut-off (B); OD values without antibody treatment (C).
virulence. Nevertheless, slime exopolysaccharide was confirmed previously as a pathogenic determinant of S. epidermidis, both in device infections as well as in infective conditions unrelated to any device.3e5,12 Its exact role as an adhesin was not previously determined. Further evidence on staphylococcal adherence on to the devices has been documented by Muller et al. who observed that a higher number of coagulase-negative staphylococci elaborating the polysaccharide adhesin (PS/A) bound to 1.5 cm segments of siliconelastomer catheters after 15 min of exposure, than did the PS/A negative isolates.13 In another similar investigation Karamanos et al. observed that antibody to the 20 kDa polysaccharide slime was immunogenic in humans and could recognise and react specifically with slime-positive S. epidermidis.10 As was evident from the present study, antibodies to slime inhibited the bacterial adherence to a significant degree, implying that slime antibody may be candidate for preventing biofilm formation in a clinical setting. To date, ours is the only study to show that an anti-slime antibody specifically inhibited adherence. Based upon our observations of a significant number of slimepositive organisms being adherent, it may be inferred that these organisms, with the potential to adhere following colonisation of devices, could trigger the pathway of slime production, intercellular
1. Gray ED, Peters G, Verstegen M, Regelmann WE. Effect of extracellular slime substance from Staphylococcus epidermidis on the human cellular immune response. Lancet 1984;1:365e367. 2. Diazo-Mitoma F, Harding GKM, Hoban DJ, Roberts RS, Low DE. Clinical significance of a test of slime production in ventriculoperitoneal shunt infections caused by coagulase negative staphylococci. J Infect Dis 1987;156:555e560. 3. Nayak N, Satpathy G, Vajpayee RB, Mrudula S. Phenotypic and plasmid pattern analysis of Staphylococcus epidermidis in bacterial keratitis. Indian J Ophthalmol 2007;55:9e13. 4. Nayak N, Satpathy G. Slime production as a virulence factor in Staphylococcus epidermidis isolated from bacterial keratitis. Indian J Med Res 2000;111:6e10. 5. Nayak N, Satpathy G, Vajpayee RB, Sethi A, Ray M, Sinha R. Clinical significance of slime production by Staphylococcus epidermidis in bacterial keratitis. Ann Ophthalmol 2002;34:204e210. 6. Arciola CR, Baldassarri L, Montanaro L. Presence of icaA and icaD genes and slime production in a collection of staphylococcal strains from catheterassociated infections. J Clin Microbiol 2001;39:2151e2156. 7. Maki DG, Weise CE, Sarafin HW. A semiquantitative culture method for identifying intravenous catheter related infections. New Engl J Med 1977; 296:1305e1309. 8. Nayak N, Satpathy G, Vajpayee RB, Pandey RM. A simple alternative method for rapid detection of slime produced by Staphylococcus epidermidis isolates in bacterial keratitis. Indian J Med Res 2001;114:169e172. 9. Arvaniti A, Karamanos NK, Dimitracopoulos G, Anastassiou ED. Isolation and characterization of a novel 20-kDa sulfated polysaccharide from the extracellular slime layer of Staphylococcus epidermidis. Arch Biochem Biophys 1994;308:432e435. 10. Karamanos NK, Syrokou A, Panagiotopoulou HS, Anastassiou ED, Dimitracopoulos G. The major 20-kDa polysaccharide of Staphylococcus epidermidis extracellular slime and its antibodies as powerful agents for detecting antibodies in blood serum and differentiating among slime positive and negative S. epidermidis and other staphylococci species. Arch Biochem Biophys 1997;342:389e395. 11. Kloos WE, Bannerman TL. Update on clinical significance of coagulase negative staphylococci. Clin Microbiol Rev 1994;7:117e140. 12. Younger JJ, Christensen GD, Bartley DL, Simmons JCH, Barrett FF. Coagulase negative staphylococci isolated from cerebrospinal fluid shunts: importance of slime production, species identification and shunt removal to clinical outcome. J Infect Dis 1987;156:548e554. 13. Muller E, Takeda S, Shiro H, Goldmann D, Pier GB. Occurrence of capsular polysaccharide/adhesin among clinical isolates of coagulase negative staphylococci. J Infect Dis 1993;168:1211e1218.
Available online at www.sciencedirect.com
Journal of Hospital Infection journal homepage: www.elsevierhealth.com/journals/jhin
Slime production is essential for the adherence of Staphylococcus epidermidis in implant-related infections N. Nayak a, G. Satpathy a, *, H.L. Nag b, P. Venkatesh c, S. Ramakrishnan d, T.C. Nag e, S. Prasad a a
Department of Ocular Microbiology, Dr Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India Department of Orthopaedic Surgery, All India Institute of Medical Sciences, New Delhi, India c Uvea & Retina Services, Dr Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India d Department of Cardiology, All India Institute of Medical Sciences, New Delhi, India e Department of Anatomy, All India Institute of Medical Sciences, New Delhi, India b
a r t i c l e i n f o
s u m m a r y
Article history: Received 20 August 2009 Accepted 10 September 2010 Available online 7 January 2011
A total of 32 Staphylococcus epidermidis isolates from indwelling device-related infections such as endophthalmitis following intraocular lens (IOL) implantation, intravenous catheter-related sepsis and orthopaedic implant infections, were studied for slime production and adherence to artificial surfaces. Of these, 21 (65.6%) isolates were slime positive by the Congo Red agar method and 24 (75%) were adherent to artificial surfaces by the quantitative slime test. The majority (19 out of 24; 79.1%) of the adherent bacteria were slime producers. Antibody to slime raised in rabbits was able to inhibit the adherence of all 24 bacteria designated as adherent by our quantitative test. It seems that slime is indispensable for the sessile mode of attachment, leading further to the development of biofilms on the indwelling devices. Ó 2010 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.
Keywords: Adherence Biofilms Device-related infections Implant-related infections Slime Staphylococcus epidermidis
Introduction
Methods
Slime is an exopolysaccharide liberated by Staphylococcus epidermidis.1 The clinical significance of slime from S. epidermidis in device-associated infections has been documented.2 Slime has also been reported as a virulence factor of S. epidermidis in bacterial keratitis.3,4 Clinical and laboratory evidence have supported the view that slime-producing S. epidermidis isolates from cases of keratitis are adherent to artificial surfaces.3,5 The molecular basis of slime production was established by amplification of the ‘ica’ locus using polymerase chain reaction assay in S. epidermidis isolates from central venous catheter-related sepsis.6 Despite all of the clinical, epidemiological, laboratory and molecular data, the exact role of slime as an adhesin has not been elucidated.2e6 The present study was undertaken to assess whether such slime exopolysaccharide produced by S. epidermidis in implant-associated infections has any direct adhesion potential, which might be involved in the process of attachment on to the indwelling implants.
Subjects and case definitions
* Corresponding author. Address: Dr Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi 110029, India. Fax: þ91 11 26588919. E-mail address: [email protected] (G. Satpathy).
Samples were collected from 73 patients with indwelling-implant related infections. They included 39 subjects of postoperative endophthalmitis following intraocular lens (IOL) implantation, 24 subjects with intravascular catheter-related sepsis, and 10 having infections due to implanted orthopaedic devices. Individuals with features of endophthalmitis such as increasing pain and redness, decreasing visual acuity, flare in the anterior chamber, corneal oedema, hypopyon, and poor glow within four to six weeks following IOL implantation were classified as cases of late-onset postoperative endophthalmitis (POE). All 39 subjects fulfilled the above criteria. The 24 patients with indwelling central venous catheters/ intravascular cannulas had clinical evidence of infection (catheterrelated sepsis) with fever 38 C, pulse rate of >90/min, respiration rate of >20/min and white blood cell count of >12 000/mm3 of blood. In addition, subjects with localised infection on the exit of the truncated tract were also considered as having catheter-related infections. Those who were having either localised inflammatory signs at the implant site or signs of sepsis, as defined above, owing to
0195-6701/$ e see front matter Ó 2010 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jhin.2010.09.023
154
N. Nayak et al. / Journal of Hospital Infection 77 (2011) 153e156
implantation of joint prostheses, nails, plates and bone cements were said to have orthopaedic implant-associated infections. Sample collection and processing Patients with POE About 0.1 mL of vitreous fluid was collected with the help of sterile tuberculin syringe and 26 gauge needle. The bevelled tip of the needle was closed with a sterile rubber bung and was transported to the laboratory immediately. The vitreous fluid was stained and cultured according to the standard procedures. Patients with intravascular implants The tips of intravascular catheters/cannulas and/or central venous catheters were collected aseptically, in sterile test tubes, and were immediately inoculated on to blood agar plates according to the rolling technique of Maki et al. and subsequently on to trypticase soy broth (TSB).7 The agar plates and TSB tubes were incubated at 37 C. For blood culture, the sample of blood was inoculated directly on to the broth. Patients with orthopaedic implants Aspirate was collected with sterile syringe and needle after properly disinfecting the surrounding skin. Pus or discharge was collected by rubbing the bed of the ulcer with a sterile cottontipped swab. If the material was insufficient, then the wound was squeezed and the exuded purulent material was collected. Intraoperative pus, if obtained, was directly inoculated on to TSB. Blood was also collected for culture if there was indication of sepsis. Additionally, the pus, aspirate, discharge were subjected to Gram staining. The material inoculated into TSB was incubated at 37 C. Growth from TSB was subcultured on to blood agar, MacConkey agar and chocolate agar plates, which were incubated at 37 C. Identification After overnight incubation, those colonies showing Grampositive cocci on smear examination were processed further. The organisms were identified and speciated according to the previously described method and were stored at e20 C as nutrient agar stab cultures until further testing.4 Slime test Test for slime production was carried out by the Congo Red agar (CRA) plate method described earlier.8
the mean OD of 10 blank cuvettes stained by the above-described procedure in that particular batch of tests.
Extraction of slime Crude slime was extracted by a procedure previously described.9 Briefly, 50 mL of mid-log phase of bacterial suspension were inoculated in 1 L of TSB and incubated for 24 h at 37 C, in humidified chambers. Extracellular material was removed from the cells by gentle shaking with glass beads in 0.15 M NaCl. The extracts were precipitated with a mixture of ethanol, sodium acetate and acetic acid in final concentrations of 80%, 0.26 M and 0.05 M respectively. The precipitate was dissolved in distilled water, the insoluble material was removed by centrifugation at 27 000 g for 30 min, and the supernatant was dialysed three times against 100 volumes of distilled water. The above procedure was repeated twice. The dialysates were centrifuged at 105 000 g for 3 h and the supernatants were freeze-dried and stored at e70 C. All of the above steps were performed at 4 C in the presence of 6-aminohexanoic acid, EDTA and phenylmethane sulphonylfluoride at final concentrations of 50, 10 and 20 mM respectively.
Antibodies to slime Rabbit antiserum to slime was prepared by immunisation of the animals by a protocol earlier adapted by Karamanos et al. with some modifications.10 Filter-sterilised antigen, i.e. crude slime, was emulsified with equal volumes of Freund’s complete adjuvant for the first injection subcutaneously and incomplete Freund’s adjuvant for the subsequent challenges one week later intravenously thrice weekly for three weeks. Animals were bled five days after final injection and antibody titres were measured by enzymelinked immunosorbent assay.
Adherence inhibition test Quartz cuvettes were coated with 1:20 dilution of the immune sera at 4 C overnight, by addition of 1 mL of diluted serum to each cuvette. Subsequent steps followed were the same as those described for the quantitative adherence test, the only exception being that bacterial suspension was added to antibody-coated cuvettes.5 The OD values recorded after such experiments (adherence inhibition test) were plotted for comparison with those recorded earlier (adherence test).
Results Quantitative slime test for adherence Adherence of each isolate to smooth surfaces was determined quantitatively by a method earlier standardised in our laboratory.5 Briefly, our procedure was as follows. Overnight cultures of bacteria in trypticase soy broth (TSB) were diluted 1 in 100 in fresh TSB, and 1 mL volume of each isolate was put into separate quartz cuvettes. After overnight incubation at 37 C, the cuvettes were washed four times with phosphate-buffered saline (PBS), fixed with Bouin’s fluid, and stained with crystal violet. Excess stain was removed by decanting the tubes and then rinsing them gently with tap water. The optical density (OD) of the stained adherent bacterial film was read with a spectrocolorimeter (Spectrocolorimeter 103, Systronics, Baroda, India) at 570 nm. Organisms were considered non-adherent if the OD was below the cut-off recorded for that batch of experiments. This cut-off was calculated as 3 SD above
Culture positivity in samples collected from patients with various implants Overall culture positivity was found in 43 (58.9%) samples, out of which S. epidermidis grew in 32 (28 as pure growth and four as mixed growth) (Table I). Other organisms recovered were: five isolates of diphtheroids from the vitreous fluid; two Pseudomonas aeruginosa, one Candida albicans and two viridans streptococci, all from intravascular catheters/cannulas; and a single isolate of Staphylococcus aureus from a pus sample in a patient with orthopaedic implant. All blood cultures were sterile. However, other organisms isolated as mixed growth along with S. epidermidis were Escherichia coli in two specimens (one from catheter and the other from pus), S. aureus in one and P. aeruginosa and E. coli in one specimen.
N. Nayak et al. / Journal of Hospital Infection 77 (2011) 153e156
155
Table I Samples collected and culture positivity among patients with various implants Implant type (no. of patients) and nature of the specimen Intraocular lens implant (39) Vitreous Cardiac implant (24) Catheter/cannula
Blood Orthopaedic implants (10) Pus/wound aspirate Blood Total a b
No. of samples
Culture positive
Culture positive for S. epidermidis alone
39
19
14
24
14
8
3a 10
Sterile 10
2b 73
Culture positive for other organisms as pure growth (no.)
Mixed growth (no.) with S. epidermidis
Diphtheroids (5)
Nil E. coli (1)
Sterile 6
P. aeruginosa (2) C. albicans (1) Viridans streptococci (2) Sterile S. aureus (1)
Sterile
Sterile
Sterile
Sterile E. coli (1) S. aureus (1) P. aeruginosa and E. coli (1) Sterile
43
28
11
4
Belong to the group of 24 cardiac patients. Belong to the group of 10 orthopaedic patients.
Phenotypic characteristics of S. epidermidis Phenotypic production of slime by all the strains was assessed by culture on CRA plates. Slime-producing strains exhibited black shiny colonies with a metallic tinge, whereas non-slime producers appeared pink (Figure 1). Of the 32 S. epidermidis isolates studied, 21 (65.6%) were slime positive (Table II). A total of 19 (90.5%) out of the 21 slime-producing organisms were adherent to artificial surfaces. By contrast, only 5 (45.4%) of the 11 non-slime producers were found to be adherent (P < 0.01). Adherence inhibition test Figure 2 shows the results of the adherence inhibition tests performed on all the 24 adherent bacteria, each plot representing an individual isolate. The middle curve denotes the cut-off OD values for the number of tests carried out at different time points.
On coating the cuvettes with slime antibody and thereafter performing the adherence test as per our protocol detailed above, there were significant diminutions in the individual OD values from those recorded without any antibody treatment (Figure 2; upper vs lower curve). This suggests that slime antibody successfully blocked the adherence of the organisms on to the artificial surfaces. Discussion During the last two decades, S. epidermidis has emerged as a major cause of nosocomial infection. This organism, which constitutes the main component of the normal skin and mucosal microflora, is particularly responsible for catheter and other medical device-related sepsis.4 Slime as a virulence factor for S. epidermidis in medical implant/device-related infections has been documented.2,11 In addition, the gene responsible for biofilm production, i.e. the ica operon, was identified in the majority of S. epidermidis strains from catheter-related sepsis, whereas none of the saprophytic strains possessed this intercellular adhesin gene.6 Despite all the above relevant documentations relating to the slime and its association with bacterial colonisation of the indwelling devices, the precise role of slime as an adhesin had not yet been established. The present study showed that 65.6% (21/32) of the strains were slime positive. By contrast, Arciola et al. reported 48.5% (33 out of 68) of their clinical isolates of S. epidermidis as slime positive.6 We also observed previously that about 43% of the corneal ulcer isolates of S. epidermidis were slime producers.3,4 The relatively high rate of slime positivity among our present isolates compared with those in our previous studies on keratitis, might suggest that S. epidermidis colonising the indwelling device may be more virulent than those colonising the corneal surface.3 Whereas Arciola et al. studied slime production on catheter isolates only; ours were not only from vascular devices, but also from others such as IOLs and orthopaedic implants.6 Thus it may be that in different microenvironments the organism behaves differently in expressing its
Table II Adherence properties of 32 S. epidermidis isolates
Figure 1. Congo Red agar test showing slime-positive bacteria with dark shiny colonies (upper left sector), slime negative bacteria showing pink-coloured colonies (upper right and lower sectors).
S. epidermidis isolates
Adherent
Slime positive Slime negative
19 5
2 6
21 11
Total
24 (75%)
8 (25%)
32
c2 ¼ 7.8, P < 0.01.
Non-adherent
Total
156
N. Nayak et al. / Journal of Hospital Infection 77 (2011) 153e156
adhesion leading eventually to the formation of biofilm, which is the rigid sessile form of the bacterial community.11,12
0.7 0.6
Conflict of interest statement None declared.
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Funding sources Supported by a grant from Indian Council of Medical Research.
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References
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20 10 OD values of tests with 24 adherent S. epidermidis isolates
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Figure 2. Adherence inhibition test result of all the 24 adherent organisms showing diminution of respective optical density (OD) values after antibody treatment (:); cut-off (B); OD values without antibody treatment (C).
virulence. Nevertheless, slime exopolysaccharide was confirmed previously as a pathogenic determinant of S. epidermidis, both in device infections as well as in infective conditions unrelated to any device.3e5,12 Its exact role as an adhesin was not previously determined. Further evidence on staphylococcal adherence on to the devices has been documented by Muller et al. who observed that a higher number of coagulase-negative staphylococci elaborating the polysaccharide adhesin (PS/A) bound to 1.5 cm segments of siliconelastomer catheters after 15 min of exposure, than did the PS/A negative isolates.13 In another similar investigation Karamanos et al. observed that antibody to the 20 kDa polysaccharide slime was immunogenic in humans and could recognise and react specifically with slime-positive S. epidermidis.10 As was evident from the present study, antibodies to slime inhibited the bacterial adherence to a significant degree, implying that slime antibody may be candidate for preventing biofilm formation in a clinical setting. To date, ours is the only study to show that an anti-slime antibody specifically inhibited adherence. Based upon our observations of a significant number of slimepositive organisms being adherent, it may be inferred that these organisms, with the potential to adhere following colonisation of devices, could trigger the pathway of slime production, intercellular
1. Gray ED, Peters G, Verstegen M, Regelmann WE. Effect of extracellular slime substance from Staphylococcus epidermidis on the human cellular immune response. Lancet 1984;1:365e367. 2. Diazo-Mitoma F, Harding GKM, Hoban DJ, Roberts RS, Low DE. Clinical significance of a test of slime production in ventriculoperitoneal shunt infections caused by coagulase negative staphylococci. J Infect Dis 1987;156:555e560. 3. Nayak N, Satpathy G, Vajpayee RB, Mrudula S. Phenotypic and plasmid pattern analysis of Staphylococcus epidermidis in bacterial keratitis. Indian J Ophthalmol 2007;55:9e13. 4. Nayak N, Satpathy G. Slime production as a virulence factor in Staphylococcus epidermidis isolated from bacterial keratitis. Indian J Med Res 2000;111:6e10. 5. Nayak N, Satpathy G, Vajpayee RB, Sethi A, Ray M, Sinha R. Clinical significance of slime production by Staphylococcus epidermidis in bacterial keratitis. Ann Ophthalmol 2002;34:204e210. 6. Arciola CR, Baldassarri L, Montanaro L. Presence of icaA and icaD genes and slime production in a collection of staphylococcal strains from catheterassociated infections. J Clin Microbiol 2001;39:2151e2156. 7. Maki DG, Weise CE, Sarafin HW. A semiquantitative culture method for identifying intravenous catheter related infections. New Engl J Med 1977; 296:1305e1309. 8. Nayak N, Satpathy G, Vajpayee RB, Pandey RM. A simple alternative method for rapid detection of slime produced by Staphylococcus epidermidis isolates in bacterial keratitis. Indian J Med Res 2001;114:169e172. 9. Arvaniti A, Karamanos NK, Dimitracopoulos G, Anastassiou ED. Isolation and characterization of a novel 20-kDa sulfated polysaccharide from the extracellular slime layer of Staphylococcus epidermidis. Arch Biochem Biophys 1994;308:432e435. 10. Karamanos NK, Syrokou A, Panagiotopoulou HS, Anastassiou ED, Dimitracopoulos G. The major 20-kDa polysaccharide of Staphylococcus epidermidis extracellular slime and its antibodies as powerful agents for detecting antibodies in blood serum and differentiating among slime positive and negative S. epidermidis and other staphylococci species. Arch Biochem Biophys 1997;342:389e395. 11. Kloos WE, Bannerman TL. Update on clinical significance of coagulase negative staphylococci. Clin Microbiol Rev 1994;7:117e140. 12. Younger JJ, Christensen GD, Bartley DL, Simmons JCH, Barrett FF. Coagulase negative staphylococci isolated from cerebrospinal fluid shunts: importance of slime production, species identification and shunt removal to clinical outcome. J Infect Dis 1987;156:548e554. 13. Muller E, Takeda S, Shiro H, Goldmann D, Pier GB. Occurrence of capsular polysaccharide/adhesin among clinical isolates of coagulase negative staphylococci. J Infect Dis 1993;168:1211e1218.