IJCRR

IJCRR - 8(11), June, 2016

Pages: 15-22

Date of Publication: 11-Jun-2016

ROLE OF BIOFILMS IN STAPHYLOCOCCUS COLONISING INTRAVENOUS CATHETERS

Author: Aruna Jadhav, Vaishali Dohe, Anju Kagal, Renu Bharadwaj

Category: Healthcare

Abstract:Introduction: Staphylococci are major nosocomial pathogen associated with indwelling medical devices, especially with intravascular catheter (I V) related infections. Major virulence factor of Staphylococcus is its ability to form biofilm on polymeric surfaces.
Objective:
• To isolate and identify the pathogens colonising IV catheters.
• To select the Staphylococcus species and to detect their ability to produce biofilms along with antimicrobial susceptibility testing.
Material and methods: A total of 373 IV catheter tips from 373 patients were collected and processed for isolation of bacterial pathogen. Total 119(31.9%) IV catheter tips were culture positive. 100 Staphylococcus strains were tested for biofilm detection by Tissue culture plate (TCP) method and Tube method (TM). Antimicrobial susceptibility testing was done by Kirby-Bauer disc diffusion method as per CLSI guidelines. The time frame since insertion and removal of catheter was also noted.
Results: 125 organisms were isolated from 119(31.9%) culture positive IV catheter tips. Staphylococcus spp.(80%) was the most common organism followed by Enterococcus spp(9% ). 93(24.4%) catheters were colonised within one week of insertion.56% of the Staphylococci were Coagulase negative staphylococci (CoNS) and 44% were S.aureus. Of 100 Staphylococcus spp. 84% were biofilm producer by TCP method and 75% by Tube method. Sensitivity and specificity of TM method vis-a-vis TCP method was 89.3%and 100% respectively.
Conclusion: Majority of Staphylococcus isolated from IV catheters were biofilm producers. TCP method is standard method for detection of strong, moderate and weak biofilm producing strains, but TM method is technically simple. Antimicrobial resistance was significantly higher in biofilm producing Staphylococcal species.

Keywords: Tissue culture plate (TCP), Tube method (TM), Biofilm, Intravascular catheter (I V).

Full Text:

INTRODUCTION
The use of indwelling medical devices is important in treatment of critically and chronically ill patients.1 These indwelling medical devices become the focus for infections, like intravascular catheter related infections. Use of vascular catheters has become an indispensable part of modern medicine practice. The predominant organisms isolated in these infections are Staphylococcus epidermidis and Staphylococcus aureus. 2 The major virulence factor for these organisms is their ability to form biofilm.2 Biofilm consist of slime which is a complex extracellular polymeric substance produced by most of the Staphylococcus. 3 In biofilm, specific initial adherence to device surface is mediated by polysaccharide adhesion (PSA). Following initial adhesion, polysaccharide intercellular adhesion (PIA) is involved in cell-cell adhesion. The synthesis of PIA is mediated by the chromosomal ica gene (intercellular adhesion), which is an operon structure contains the icaADBC genes.4,5 Biofilm formation is a major concern in nosocomial infections because it protects microorganisms from host immune response along with antimicrobial agents. Such infections are resistant to systemic antibiotic therapy and removal of infected device becomes necessary. Therefore, once biofilmassociated Staphylococcal infections occur, they are difficult to eradicate.6 Biofilm formation in Staphylococcus can be studied by various methods such as-microscopic examination by using epifluroscence, scanning electron microscope (SEM), confocal laser scanning microscope (CLSM). Molecular techniques such as Polymerase chain reaction (PCR) which amplifies the gene (ica ABCD).7 The present study was carried out to assess the incidence of Staphylococcus spp. colonising intravenous catheters. An effort was also made to study the presence of biofilm in these Staphylococcus spp and their antimicrobial sensitivity pattern. MATERIAL AND METHODS: Collection and processing of IV catheter tips: A total of 373 IV catheter tips (336 peripheral venous catheter tips, and 37 central venous catheter tips) were collected from 373 patients, admitted in Medicine, Surgery, Paediatrics wards, and Intensive care units. The distal 5 cm of catheter was cut off and placed in a sterile screw caped container with 1ml brain heart infusion (BHI) broth.10 [fig.1] All tips were processed for quantitative culture technique. For this, catheter tip in BHI broth was vortexes for 1min and 0.1 ml of suspension was spread over Blood agar and MacConkey agar 11,12. The plates were incubated aerobically overnight at 370 C and were observed for growth. Catheter tip colonization was said to be present if more than 100 cfu /catheter segment by this vortexing techniques. 11 A total of 100 Staphylococcus spp. were isolated from 373 catheter tips. Speciation was done by Standard microbiological techniques. 11 All Staphylococcus isolates were subjected to antimicrobial susceptibility testing on Muller-Hinton agar by Kirby-Bauer disc diffusion method14. Methicillin resistance was detected by using Cefoxitin-30μg disc as per CLSI.14Inducible Clindamycin resistance were detected by D test as per CLSI guidelines. 14A record was kept of the time since insertion of all catheters removed and cultured in the present study. Biofilm detection methods: Biofilm formation in Staphylococcus spp. was done by tube method(TM) and tissue culture method (TCP).This was done to evaluate the usefulness of the tube method for biofilm detection. Tube method(TM):1,3 10 ml Trypticase soya broth (TSB) with 1% glucose was taken in a new glass test tube which was inoculated with loopful of Staphylococcus growth from overnight culture plate. It was further incubated at 37°C for 24 hours. Tube was decanted and washed with PBS (PH 7.3) and then further it was air dried. A dried tube was stained with 0.1% crystal violet. Excess stain was removed by distilled water. Tubes were dried in inverted position and observed visually for biofilm formation. Each test was performed in triplicate to minimise errors. S. epidermidis A TCC 35984 (strong biofilm producer) used as positive control while, uninoculated TSB broth with 1% glucose as negative Control. Biofilm formation is said to be positive when a visible thin film lined the wall and bottom of the test tube. Ring formation at the liquid interface is not considered as an indication of biofilm formation. Biofilm formation was scored asStrong, moderate, weak, and negative. [fig.3] Tissue culture plate method (TCP): 1,9,15 A. Biofilm Cultivation - All Staphylococcal isolates from fresh agar plates were inoculated in TSB with 1% glucose and incubated for 18 hr at 37o C in stationary condition and then diluted 1 in100 with fresh medium. Individual wells of sterile, polystyrene, 96 well-flat bottom tissue culture plates were filled with 200µL aliquots of the diluted cultures. The inoculated plate was covered with a lid and incubated aerobically for 24 hr at 370 C under static condition. B. Washing - After incubation content of each well was gently removed by tapping the plates. Wells were washed three times with 300 µL of sterile phosphate-buffered saline (PBS; PH 7.2). C. Fixation - After washing remaining attached bacteria were fixed by air drying. D. Staining - Adherent biofilm layer formed in each microtiter plate well was stained with 150 µL of 0.1% Crystal violet for 15 min at room temperature. After staining, washing was done until washing are free of stain. Then microtiter plate was air dried at room temperature. S. epidermidis ATCC 35984 (strong biofilm producer) used as a positive control while uninoculated TSB broth with 1% glucose as negative control. E. Measurement and interpretation of results15 Uniformly Stained adherent Staphylococcal cells on well of microtiter plate were considered as biofilm producer [fig.3].

Optical density (OD) of the stained adhesive Staphylococcus bacteria was determined with a micro-ELISA reader at a wavelength of 570 nm (OD570 nm ). The result of the test is recorded and these OD values are considered as a true indication of bacteria adhering to the surface and biofilm formation. Tests were performed in triplicate to minimise errors and for analysis of data. Interpretation of obtained results requires definition of the cut –off value that separates biofilm producing from nonbiofilm producing strain. i. Average OD values were calculated for all tested strain (ODt) and negative controls (since all tests are performed in three times). ii. Cut-off value (ODc) was calculated. It is defined “as three standard deviations (SD) above the mean OD of negative value”. [ODc = average OD of negative control + (3xSD of negative control)]. iii. ODc value was calculated for each microtiter plate separately. For easier interpretation of results, strains were divided in to the following categories. • No biofilm producer = ODt ≤ ODc • Weak biofilm producer = ODc < ODt ≤ 2xODc • Moderate biofilm producer = 2xODc < ODt ≤ 4x ODc • Strong biofilm producer = 4x ODc < ODt Statistical Methods: Statistical evaluation of the TM method for detection of biofilm formationResults and observations were analysed by using In Silico statistical software. Chi-square test and P-value was calculated by using this software for comparison and to determine the statistical significance Results and observations: Total 373 IV catheter tips were processed out of which 119 (31.9%) were culture positive, which include 95(28.3%) peripheral and 24(64.9%) central IV catheter tips.[Table1] Common indication for peripheral IV line removal [85 i.e.89.5%] was when no longer required. In central IV line it was removed in majority of patients [20 i.e.83.4%] due to local sign and symptoms of catheter related infection. Peripheral IV lines were colonised with bacteria earlier than central IV line.55 (57.9%) peripheral IV catheter colonization was seen after 2-4 days of catheter use while100% colonisation with CVC when duration of catheter use was > 7days [Table 2] Total 125 organisms were isolated from 119 culture positive IV catheter tips. Staphylococcus spp.100 (80%) was the most common organism followed by Enterococcus spp 9%.[Table 3] 6 catheter tips showed mix growth i.e. two organism from each. Out of 100 Staphylococcus species, 44% were S.aureus and 56% CoNS. Amongst the all CoNS, S.epidermidis 22 (39.28%) was the commonest isolate followed by S.haemolyticus 13 (23.1%). [Table 4] In present study 84% of Staphylococci were biofilm producer by tissue culture plate method and 75% by tube method. TCP method is considered standard test for biofilm detection. The tube method is relatively and technically simple method. Out of these 84% of Staphylococci 39(46.4%) were S.aureus and 45(53.3%) were CoNS. However in CoNS 21(52.5%) were S.epidermidis and 10(25 %) S.haemolyticus 3] 6 catheter tips showed mix growth i.e. two organism from each. Out of 100 Staphylococcus species, 44% were S.aureus and 56% CoNS. Amongst the all CoNS, S.epidermidis 22 (39.28%) was the commonest isolate followed by S.haemolyticus 13 (23.1%). [Table 4] In present study 84% of Staphylococci were biofilm producer by tissue culture plate method and 75% by tube method. TCP method is considered standard test for biofilm detection. The tube method is relatively and technically simple method. Out of these 84% of Staphylococci 39(46.4%) were S.aureus and 45(53.3%) were CoNS. However in CoNS 21(52.5%) were S.epidermidis and 10(25 %) S.haemolyticus. Sensitivity and specificity of TM method vis-a-vis TCP method was 89.3%and 100% respectively with 100% Positive predictive value (PPV), and 64% negative predictive value (NPV). Biofilm formation by tube method was found to be equivalent in specificity to the tissue culture plate method for detecting biofilms. [Table 5] TM method was comparable with TCP method to differentiate strong and moderate biofilm producing strains but not the weak one. The TCP method was definitely better in detecting weak biofilm producer. [Table 6] In this study >50% Staphylococcal spp. were multi drug resistant (MDR) [Table-7]. Overall 75% of S. aureus and 60.7% CoNS were resistant to Penicillin and Methicillin resistance was 63.6% in S. aureus and 42.8% in CoNS [Table7]. However, inducible Clindamycin resistance (MLSBi) was 21% amongst all Staphylococcus species and constitutive Clindamycin resistance (MLSBc) was 35%. Antimicrobial resistance was higher in biofilm producing Staphyloccus spp. than non biofilm forming one [Table 8].

DISCUSSION
The extensive use of intravenous catheters in hospitalized patient has led to increased incidence of catheter-related infection (CRI), especially blood stream infections. These infections originate from the microbial colonisation of the intravascular catheters. 16 In the present study out of 373 IV catheter tips processed, 119 (31.9%) were culture positive [Table 1]. Rao SD et al (2005) reported 74(54.8%) culture positive out of total 135 IV catheter tips. This was higher than present study. This could be probably due to the fact that IV catheter tips were collected from paediatric intensive care unit (PICU) only in theirstudy.17

Peripheral IV catheter tip culture positivity rate, in our study was 95 (28.3%). In Nahirya P et al (2008) showed 20.7%

culture positivity of peripheral IV catheter tips collected from paediatric wards.18However, Rao SD et al (2005) detected 54(52.4%) were culture positive IV tips out of 103 peripheral IV catheters.17 Central IV catheter (CVC) tip culture positivity rate in present study was 24 (64.9%) This was comparable with 62.5% each given by Subba Rao SD et al in (2005)17 and Gahlot R et al in (2013).19While Chopdekar K et al(2011) found that culture positivity rate was 57.6% in their study. 20 Peripheral IV catheters were removed in 89.5% of patients when they were no longer required. However, Central IV catheter removal in 83.4% of patients was due to the presence of clinical sign and symptoms of infections like local signs of inflammation .Majority of CVC catheter tips were collected from adult ICUs with co-morbid conditions like hypertension, diabetes, malignancy or receiving chemotherapy. The duration of IV catheterisation is a significant factor which determined the development of catheter related infections. In our study peripheral IV catheter colonisation was seen, in 57.9% of patients, after 2-4 days of catheter use [Table2]. These findings were comparable with Rao SD et al17 (2005). In present study central IV catheters culture positivity rate was 100% when duration of use was more than7days [Table2]. Rao SD et al (2005) showed 100% culture positive rate, after duration more than 11days. 17 Total 125 organisms were isolated from 119 cultures positive catheter tips in our study. Out of which CoNS 44(44.8%) and S.aureus 56(35.2%) were predominant ones, followed by Enterococcus spp.(9%)[Table 3]. Nahirya et al (2008) found S.aureus (60.5%) as the predominant pathogen followed by S.epidermidis (23.4%).18 Rao SD et al (2005)17 detected CoNS (32.4%) followed by Pseudomonas spp.(31%).While in Chopdekar et al, (2011) study revealed maximum colonisation with non-albicans Candida spp.(22.6%).20The microbial profiles of catheter colonisation vary in different settings or areas due to the impact of environmental contaminants in the pathogenesis of device related infections. CoNS is frequently responsible for catheter colonization due to its capacity to adhere to polymer surfaces and consequent biofilm production. Out of 100 Staphylococcus spp. isolated in our study, 56% were CoNS and 44% were S.aureus. Two recent studies Prasad S et al21 (2012) and Patil HV et al (2011) isolated 57.1% and 65% of CoNS respectively from indwelling IV catheter tips, which were comparable with our study.22 Amongst the 56% CoNS in present study 39.28% were S.epidermidis followed by S.hemolyticus 23.1% [Table 4]. Patil HV et al (2011)22 showed 45% of S.epidermidis and 15% of S.hemolyticus in their study. S.aureus is a known pathogen in hospital infections. It was the second common organism in our study which was (35.2%). Rate of S.aureus was much lower in Khanna et al (2013) 23 who got 13.25% as compared to our study The higher rate of S.aureus in present study could be due to the lack of dedicated IV catheter insertion team, as well as lack of standardized protocol for insertion and replacement of IV catheters. In the present study 100 Staphylococcus spp. were screened for biofilm detection by modified TCP method and TM method, along with antimicrobial susceptibility pattern. 84% and 75% of Staphylococcus spp. showed biofilm production by TCP and TM method respectively [Table 5].Bose et al (2009) reported 54.2% and 42.4% of Staphylococcus spp. were biofilm producer by TCP and TM method respectively.24 While Mathur et al (2006) showed 53.9% and 41.4% of Staphylococcus spp. as biofilm producer by TCP and TM method respectively.1 Biofilm production in present study was higher than these studies. These could be because the isolates from above two studies were obtained from all clinical samples and not only catheter tips like the current study. In this study 84% Staphylococcus species were true biofilm producer, i.e. positive by standard TCP method [Table-5]. However, 9 strains were positive by TCP but negative by TM method. This is because it was difficult to differentiate between weak and non-biofilm producers by TM method. Otherwise TM method correlated well with TCP method for Strong and moderate biofilm production.[Table-6]. TCP method was definitely better test to detect weak biofilm producing strains. This variability in results of weak biofilm production was also observed by other similar studies.1,24 Sensitivity and specificity of TM method vis-a-vis TCP method was 89.3% and 100% respectively with 100% Positive predictive value (PPV), and 64% negative predictive value (NPV) [Table-5]. This finding is supported by two similar studies. 1,24 Multidrug resistance (MDR) was significantly higher in all biofilm producing strains than non biofilm producing strains in present study [Table-8]. MDR strain was taken as a strain resistant to > 2 classes of antibiotic. Thus making it difficult to treat intravenous catheter related infections. There are some highly accurate methods like PCR to detect icaADBC operon. These encode polysaccharide intercellular adhesion (PIA) which mediates biofilm formation. These are expensive methods. In a developing country like India, low cost method like TCP and TM methods are useful for screening purpose of biofilm producing strains.

CONCLUSION
To conclude, present study showed biofilm formation in 84% of the Staphylococcus species isolated from IV catheters tips.

Since this colonisation will result in blood stream infections unless the catheter is removed or changed early. Simple preventive measures, such as aseptic precaution during catheter insertion, daily catheter care, monitoring of catheterised patients, could help to reduce risk of colonisation and subsequent catheter related infections. Since these infections are difficult to treat, it is better to prevent such infections than attempt to treat, once they are established.

ACKNOWLEDGEMENT
I (Author) take this opportunity to acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The Authors are also grateful to authors/ Editors/ publishers/ of all those articles, journals and books from where the literature for this article has been reviewed and discussed. Ethical clearance- Not Applicable Informed consent- Not Applicable Source of funding- Not Applicable Conflict of interest- Not Applicable

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