Environmental regulation of biofilm formation in intensive care unit isolates of Staphylococcus epidermidis

Journal of Hospital Infection (2002) 42: 212±218 doi:10.1053/jhin.2002.1309, available online at http://www.idealibrary.com on

Environmental regulation of biofilm formation in intensive care unit isolates of Staphylococcus epidermidis F. Fitzpatrick*y, H. Humphreys*y, E. Smythy, C. A. Kennedy* and J. P. O'Gara* *Department of Microbiology, RCSI Education and Research Centre, Smurfit Building, Royal College of Surgeons in Ireland, Dubliq, Ireland and yDepartment of Microbiology, Beaumont Hospital, Dublin, Ireland Summary: Staphylococcus epidermidis is a common cause of prosthetic device-related infection in the intensive care unit (ICU). The environmentally regulated ica operon encodes a polysaccharide adhesin which is a key virulence determinant in the development of S. epidermidis biofilms. To evaluate the capacity of ICU S. epidermidis isolates to form biofilm, we measured biofilm production by 18 isolates associated with devicerelated infection and 20 contaminating isolates that were not associated with clinically diagnosed infection. Biofilm assays were performed in brain±heart infusion (BHI) medium and in BHI supplemented with salt, ethanol or subinhibitory tetracycline, all of which have the potential to promote biofilm formation. Polymerase chain reaction (PCR) was used to screen for the presence of the ica genes. A significant proportion of S. epidermidis strains associated with device-related infections (89%) were found to contain the ica locus compared with 50% of contaminating isolates (P ˆ 0.01). However only four of 26 (15.3%) of all ica-positive isolates were biofilm-positive when grown in BHI medium, indicating that no significant association existed between the presence of the ica locus and biofilm-forming capacity, under standard growth conditions. In contrast the number of ica-positive isolates that were biofilm-positive under stress-inducing growth conditions or in the presence of subinhibitory tetracycline increased significantly to 73% (P ˆ 0.02). These findings suggest that the presence of the ica locus alone is not sufficient for biofilm formation and that regulation of biofilm formation under altered growth conditions, which may exist in the in vivo environment, also plays a possible role in the pathogenesis of biomaterial-related S. epidermidis infections. & 2002 The Hospital Infection Society

Keywords: Biofilm formation; ica operon; environmental regulation; clinical isolates. Introduction Coagulase-negative staphylococci (CoNS) are among the most common causes of hospital-acquired

Received 2 May 2002; revised manuscript accepted 2 August 2002. Aspects of this study have been submitted for presentation at the 5th International Conference of the Hospital Infection Society scheduled for September 2002. Author for correspondence: Dr J. P. O'Gara, Department of Microbiology, RCSI Education and Research Centre, Smurfit Building, Royal College of Surgeons in Ireland, Dublin 9, Ireland. Tel.: 353-1-809 3711; Fax: 353-1-809 3709; E-mail: [email protected]

0195±6701/02/110212 ‡ 07 $35.00/0

infection in the intensive care unit (ICU).1 The clinical presentation of staphylococcal infection is not unique but their treatment is increasingly problematic because of increasing resistance to a widening spectrum of antimicrobial agents. ICU patients have multiple risk factors for staphylococcal infection, partly because of their serious underlying disease, and also because of exposure to multiple invasive procedures, implanted prosthetic devices and vascular grafts. Most staphylococcal infections result in acute disease, however bacterial persistence and recurrent infections are also observed, particularly in patients with indwelling medical devices.2 & 2002 The Hospital Infection Society

Biofilm formation in S. epidermidis isolates

The majority of biomaterial infections are caused by staphylococci. S. epidermidis and other CoNS cause between 50±70% of catheter-related infections,3 48±67% of infective complications following central nervous system shunt procedures,4 40±50% of prosthetic cardiac valve infections5 and up to 50% of joint replacement infections.6 The high rate of intravascular catheterization among hospitalized patients, and in particular ICU patients, highlights the clinical impact of these infections. Production of CoNS biofilm, which is composed of sessile bacterial cells embedded in a protective extracellular polysaccharide matrix, is widely considered to be an important pathogenic determinant in prosthetic device-related infections. Once formed, biofilms are resistant to antimicrobial chemotherapy and host immune responses, and can be very difficult to treat clinically. A number of reports have suggested a potential correlation between levels of resistance to -lactam antibiotics and the biofilm phenotype in staphylococci.7±10 Persistent infection of a prosthetic implant can serve as a septic focus, especially if caused by S. aureus, which can result in seeding to other organs (endocarditis, osteomyelitis, brain abscess), resulting in increased morbidity and mortality. Formation of S. epidermidis biofilms is thought to occur in a two-step manner.11±15 A cellular accumulation process to form the mature biofilm follows a rapid initial attachment to an inert plastic surface. At the biochemical level extracellular polysaccharides play an important role in biofilm formation. Capsular polysaccharide adhesin (PS/A)12 and/or one of several proteins (including autolysin16) have been proposed to mediate initial adherence of S. epidermidis, while accumulation of cells is due to production of polysaccharide intercellular adhesin (PIA).13 The PIA is encoded by the ica (intercellular adhesin) operon,13 however recent reports suggest that this operon also encodes PS/A and that PS/A and PIA are chemically related.17 The icaADBC operon encodes the enzymes required for PIA biosynthesis13,18 and a number of groups have isolated S. epidermidis biofilm-negative strains with transposon insertions mapping to the ica locus.12,13,19,20 The ica gene cluster appears to have an important role in the pathogenesis of S. epidermidis infections. In one study approximately 85% of S. epidermidis blood culture isolates were found to contain the ica genes compared with 6% of saprophytic isolates,21 while a number of other studies have indicated that the ica locus is an important marker discriminating between significant and contaminating isolates.7,22

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Animal models have also pointed to the importance of the ica operon and polysaccharide adhesin in biomaterial infections.23±25 Expression of the ica operon is regulated by environmental parameters in the laboratory and also under in vivo conditions. Anaerobic growth,26 the presence of sub-inhibitory concentrations of certain antibiotics,27 and osmotic stress28 all result in elevated expression of the ica operon or PIA synthesis. Importantly while few clinical isolates of S. aureus produce polysaccharide adhesin in vitro it is elaborated during human and animal infection,29 an observation that may also be true for S. epidermidis.27,30 In this study we evaluated the ica status and biofilm-forming capacity of S. epidermidis isolates associated with device-related infections in acutely ill ICU patients under standard and biofilm-inducing growth conditions. For control purposes, these findings were contrasted with the evaluation of S. epidermidis isolates from ICU patients who were not clinically infected. Materials and methods Beaumont Hospital, Dublin, is a 650-bed tertiary referral teaching hospital, containing the national renal transplantation and neurosurgical units in the Republic of Ireland. The hospital incorporates an 11 bedded general ICU and a 10 bedded neurosurgical ICU. The microbiology team conduct daily ICU rounds with the ICU team where the relevance of isolates from the laboratory and other problems such as the use of antibiotics are discussed. Collection and identification of isolates CoNS isolates from cerebrospinal fluid (CSF), blood cultures and implanted medical devices were collected prospectively in the ICU from 1 August 2001 to 30 November 2001. Isolates were stored at ÿ80 C on Protect beads (Technical Service Consultants Ltd, Lancashire) within 24 h of being cultured from appropriate specimens. Isolates were revived from Protect beads onto brain±heart infusion agar (BHIA, Oxoid Ltd, UK) and streaked for single colonies to determine purity. Presumptive CoNS were identified on the basis of a positive catalase test, negative coagulase and negative DNase tests. CoNS were screened by polymerase chain reaction (PCR) to identify S. epidermidis strains using specific

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oligonucleotide primers, as previously described.31 Methicillin susceptibility testing was performed using a modified Stokes' method.32 The wellcharacterized, biofilm-forming reference strain S. epidermidis ATCC 35984 (RP62A)33,34 was used as a positive control for PCR reactions and the detection of biofilm production.

Patient data and the clinical interpretation of isolates Patient demographics, details of antibiotics prescribed, type of implanted devices present, clinical significance and antibiotic sensitivity pattern of each isolate were also collected prospectively and stored on a database (Microsoft Access). The relevance of isolates was discussed on a daily basis with the ICU team, and assigned as either clinically significant isolates or probable contaminants. Line sepsis was defined as (1) isolation of a staphylococcus from a line tip in a patient with the same isolate in blood cultures and a clinical picture of sepsis with no alternative source or (2) isolation of a staphylococcus from a line tip or blood culture in a patient with lines in situ, with a clinical picture of sepsis with no alternative source and where sepsis improved on line removal and/or treatment with anti-staphylococcal antibiotics. Ventriculoperitoneal (VP) shunt infection was defined as isolation of a staphylococcus from one or more CSF or external ventricular drain (EVD) cultures, in a patient with a VP shunt present and a clinical picture of sepsis with no alternative source.

Genetic techniques MWG Biotech (Germany) supplied all oligonucleotide primers used for PCR. Two sets of primers, ICAR1 (5 0 -CTCGAATTTGTTACATACTAG3 0 ) and IPR2 (5 0 -TTGGAT-AGAAAAGTAAAAAG-3 0 ) and IPR1 (5 0 -GCGTTATCAATAATCTTATC-3 0 ) and ICAD1 (5 0 -GATATTGGTGTTGCATTCGTC-3 0 ) were used in PCR reactions to screen for the presence of the ica locus in S. epidermidis isolates under the following conditions 94 C 20 s, 50 C 20 s and 72 C 20 s for 30 cycles. PCR reactions using S. epidermidis cells taken from colonies grown on BHIA as template proved efficient, reproducible and more rapid than extraction of total DNA prior to amplification.

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Detection of biofilm production Quantitative determinations of biofilm formation in 96-well tissue-culture plates (Sigma, UK) were performed based on the method of Christensen et al.35 as described by Ziebuhr et al.21 with the following modifications. Duplicate assays were performed for each strain grown in eight individual plate wells in BHI (Oxoid, UK), BHI supplemented with NaCl (4%) and glucose (0.5%), BHI supplemented with ethanol (4%) and BHI supplemented with tetracycline (0.06 mg/mL) at 37 C for 24 h. Plates were subsequently washed three times with distilled water and dried for 1 h at 56 C as recommended by Gelosia et al.36 prior to staining with a 0.4% crystal violet solution. The absorbance of the adhered, stained cells was measured at 592 nm using a Multiskan plate reader, Flow Laboratories, UK. Absorbance readings of >0.12 were considered biofilmpositive and readings of <0.12 were recorded as biofilm-negative. Statistical analysis Variables were compared between groups using the Chi-square test. All analyses were performed using the S.P.S.S. statistics package for Windows. Results Collection of isolates Thirty-eight S. epidermidis isolates were collected over the study period. All patients were ventilated and had a minimum of two intravenous lines and one peripheral arterial line present when samples were taken. Source of isolates, methicillin sensitivity, ica status and clinical diagnosis after discussion on the ICU round are outlined in Table I. Eighteen (47%) of the isolates were associated with implanted biomaterial infections and the majority of patients, 34, were in hospital greater than one week. Clinical significance, presence of ica and biofilm formation S. epidermidis isolates were screened for the presence of the ica operon using direct PCR on cells taken from colonies, as described in methods. There was a statistically significant association between a device infection-associated S. epidermidis isolate and the presence of the ica gene cluster. Sixteen of 18 (89%)

Biofilm formation in S. epidermidis isolates

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isolates associated with IV line- or VP shunt-related infections and 10 of 20 (50%) contaminating isolates were identified as positive for the ica operon (P ˆ 0.010) (Table I). Quantitative determinations of biofilm formation were made by measuring adherence of broth cultures to 96-well tissue culture plates as outlined in Materials and Methods. Under standard growth conditions in BHI broth only three of 18 (16%) device infection-associated S. epidermidis and 1/20 (5%) contaminating S. epidermidis isolates were found to be biofilm forming. To evaluate the impact of environmental growth conditions on biofilm formation by clinical isolates we performed biofilm assays using growth media supplemented with Table I Details of ICU S. epidermidis isolates Clinically Significant*

Total Clinical diagnosisy IV infection VPS infection Source Blood Line tip CSF EVD tip VPS Methicillin susceptibility Sensitive Resistant Quantitative determination of biofilm formationz BHI Induced

Contaminant*

Total

ica ‡

ica ÿ

ica ‡

ica ÿ

38

16

2

10

10

8 10

6 10

2 Ð

Ð Ð

Ð Ð

18 4 9 6 1

3 3 8 1 1

2 Ð Ð Ð Ð

8 Ð Ð 2 Ð

5 1 1 3 Ð

7 31

2 14

1 1

1 9

3 7

4 23

3 11

Ð Ð

1 8

Ð 4

* ica ‡ , ica gene cluster present; ica ÿ , ica gene cluster absent. y IV, intravascular; VPS, ventricular peritoneal shunt; EVD, external ventricular drain. z BHI, biofilm formation in brain±heart broth; induced, biofilm formation induced altered growth conditions (i.e., in the presence of NaCl, EtOH or subinhibitory tetracycline concentrations).

NaCl/glucose, ethanol or sub-inhibitory concentrations of tetracycline, as described previously.27,28 This resulted in an increase in the numbers of isolates capable of biofilm formation, with 11 of 18 (61%) of device-related and 12 of 20 (60%) contaminating isolates producing biofilm in the presence of one or more of these media supplements (Table I). NaCl was the most effective biofilm-promoting stimulus and enhanced biofilm production in 50% of the isolates. Individual clinical isolates had different responses to the biofilm-promoting growth conditions examined and strains could be placed in seven groups on the basis of this differential regulation of biofilm formation (Table II). The relationship between the presence of the ica gene cluster in an isolate and biofilm production was also investigated. Although there was no significant association between presence of ica and biofilm formation in BHI (P ˆ 0.151), there was a statistically significant association between the presence of ica and the ability to form biofilm under biofilm-stimulating environmental conditions (P ˆ 0.02) (Table I). Methicillin susceptibility and biofilm production The majority of isolates examined in this study were methicillin resistant (Table I). Although not statistically significant, a higher proportion of methicillinresistant S. epidermidis isolates were positive for ica (N ˆ 31, 74%) than methicillin-sensitive isolates (N ˆ 7, 43%) (P ˆ 0.124). However there was no association between methicillin resistance in these isolates and their capacity to produce biofilm under standard laboratory conditions (P ˆ 0.815) or under biofilm-inducing conditions (P ˆ 0.697). Discussion In the last two decades, with the increased use of prosthetic biomedical implants, nosocomial infections caused by Gram-positive bacteria, in particular staphylococci, have become more prevalent as a cause

Table II Differential regulation of biofilm production in ICU S. epidermidis isolates under biofilm-inducing growth conditions Isolate

Clinically significant Contaminant

Biofilm induction by media supplements* NaCl/G only

EtOH only

Tet only

NaCl/G and EtOH

NaCl/G and Tet

EtOH and Tet

NaCl/G, EtOH and Tet

1 7

1 1

1 Ð

5 1

1 2

1 Ð

1 1

* Biofilm formation was measured after overnight growth at 37 C in brain±heart infusion broth supplemented with Tet (sub-inhibitory concentration, 0.06 mg/mL, of tetracycline), NaCl/G (4% NaCl and 0.5% glucose), EtOH (4% ethanol).

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of hospital-acquired infection. In this study we focused on S. epidermidis strains associated with implanted biomaterial infections, specifically VP shunt and IV line infections in ICU patients. All isolates were clinically defined in terms of their symptoms and stored fresh for subsequent study. Such infections are likely to involve staphylococcal biofilm formation on the device surface. Although the total number is relatively small, these isolates were well characterized both clinically and in the laboratory, and we believe that they are representative of the total S. epidermidis population in our ICUs. Consistent with previous studies, our data revealed a significant correlation between clinical significance, particularly in device-associated S. epidermidis isolates, and the presence of the ica genes. However 55% of contaminating S. epidermidis strains were also ica-positive. This may be explained in part by the fact that the contaminating strains in this study were isolated from patients who had been in the ICU for at least one week and may indicate that ica-positive S. epidermidis strains are prevalent in the ICU environment. A recent study found no significant difference in ica status between diseaseassociated and contaminating S. epidermidis strains, all of which were isolated from a neonatal ICU.37 Surprisingly, only a small proportion of the icapositive S. epidermidis strains implicated in devicerelated infection produced biofilm under standard growth conditions (N ˆ 16, 18.7%). However, when grown under stress-inducing conditions or in the presence of sub-inhibitory tetracycline, the proportion of these isolates which produced biofilm increased to 69% (Table I). These findings indicate that the capacity to form biofilm is a regulated phenotype, and that environmental conditions, such as stress or subinhibitory antibiotic concentrations, may promote biofilm formation on implanted medical devices. Interestingly we also observed a similar trend among ica-positive contaminating S. epidermidis strains where the proportion of biofilm-positive clinical isolates increased from one of 10 (10%) to eight of 10 (80%) following growth under biofilm inducing conditions. These data suggest that ica-positive S. epidermidis strains circulating in the ICU are more likely to cause device-related infections in which the control of the principal pathogenic mechanism, namely biofilm production, is complex. These conclusions are consistent with those of the neonatal ICU study that found that quantitative biofilm production was greater in isolates

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from neonates with bacteraeamia.37 The authors suggested that regulation of biofilm expression might play a central role in bacteraemia caused by CoNS. The recent report that alcoholic ingredients in skin disinfectants have the potential to increase biofilm formation in ica-positive S. epidermidis clinical isolates also suggests that biofilm regulation may contribute to the development of foreign bodyrelated infections.38 Our observations suggest that both biofilm regulation and the presence of the ica locus in S. epidermidis ICU isolates may play an important role in the pathogenesis of biomaterialrelated infections but that the response of individual isolates to biofilm-promoting growth supplements varies (Table II). These divergent biofilm responses suggest that S. epidermidis strains in the ICU have the potential to form a biofilm but that the capacity of individual strains to cause disease may also be influenced by host factors and environmental conditions at the site of infection. Biofilm production was also induced in a number of isolates lacking the ica gene cluster indicating that in some staphylococcal strains an alternative mechanism can be employed to facilitate biofilm formation. The biofilm-inducing stimuli (NaCl, ethanol, sub-inhibitory tetracycline) tested in this study have been shown to induce ica operon transcription or PIA production,27,28 but may also promote biofilm formation by an ica-independent mechanism. Previous studies have suggested a possible association between resistance to -lactam antibiotics and the ability to form biofilm in staphylococci.7±10 As would be predicted from length of stay data, where 89% of isolates were from patients in hospital for more than a week, 81% of staphylococcal isolates collected in this study were methicillin resistant (Table I). An association between the presence of ica and methicillin resistance in these ICU isolates was on the borderline of being statistically significant, perhaps due to insufficient numbers of isolates. However there was no significant association between the capacity of these S. epidermidis isolates to produce biofilm in vitro (under standard and biofilm-promoting growth conditions) and their susceptibility to methicillin. These data may suggest that these virulence determinants are independent and confer specific selective advantages for persistence and colonization, both of which are likely to be critical for pathogen survival in the ICU environment. In conclusion, this study suggests that the ica locus is a virulence marker in S. epidermidis ICU

Biofilm formation in S. epidermidis isolates

strains associated with clinically-diagnosed infections but that the production of biofilm by icapositive ICU strains is also dependent upon environmental conditions. This environmental regulation is complex and the host environment and site of infection may also play an important role in the pathogenesis of biomaterial-related disease caused by S. epidermidis. Acknowledgements F.F. was supported by MRC (Path) Project Grants from the Hospital Infection Society and from the Association of Medical Microbiologists. C.A.K. is funded by a grant from Pfizer Ltd (Ireland) to the Department of Microbiology, Royal College of Surgeons in Ireland. We would like to thank Mr Peadar Clarke for his support and advice and Ms Diana Katiman who conducted preliminary experiments for this study.

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