Basher, Farhana, Alim, and Khan: Detection of catheter related blood stream infections in an ICU of a tertiary care center in Northern India


Introduction

Microorganisms produce extracellular polysaccharides, resulting in the formation of a biofilm that attach to surfaces. Because of the increased resistance of biofilm-associated organisms to antimicrobial agents they pose a serious problem for public health There is a potential for these organisms to cause infections in patients with indwelling medical devices. Many bloodstream infections and urinary tract infections are associated with indwelling medical devices and, therefore, are (in most cases) biofilm associated. The most effective strategy for treating these infections may be removal of the biofilm contaminated device.1

A biofilm is defined as an architectural colony of microorganisms, within a matrix of extracellular polymeric substance that they produce. Biofilm contains microbial cells adherent to one-another and to a static surface (living or non-living). Biofilms are usually pathogenic in nature and are associated with nosocomial infections. The National Institutes of Health (NIH) has shown that 65% of microbial and 80% of chronic infections are associated with biofilm formation. There are many steps in biofilm formation, starting with attachment to a living or non-living surface that will lead to formation of micro-colony, giving rise to three-dimensional structures and ending up, after maturation, with detachment. Several species of bacteria communicate with one another during biofilm formation, through quorum sensing. In general, bacterial biofilms show resistance against human immune system, as well as against antibiotics. There are great health related concerns because of biofilm potential to cause diseases, utilizing both device-related and non-device-related infections.2

Most hospitalized patients have an intravenous catheter, and up to 30% have a central venous catheter for hemodynamic support, for infusion of medications that require instillation into a central vein, or for long-term venous access.3 These catheters are a major source of health care-associated infections. The Centers for Disease Control and Prevention estimated that 18,000 central lines associated blood stream infections occurred among patients in U.S. intensive care units and 23,000 among patients in inpatient wards in 2009.4

Formation of biofilm is universal on central venous catheters,but the location and extent of biofilm formation depend on the duration of catheterization. A short term (less than 10 days) catheters have more biofilm formation on external surface,whereas long term catheters (30 days) have greater biofilm formation on catheter lumen. Growth of microbes maybe affected by the nature of the fluid which is administered through central venous catheter.5, 6 Both Gram negative and Gram positive bacteria can form biofilms on indwelling medical devices. The most common biofilm forming bacteria are Enterococcus faecalis, Staphylococcus aureus, S. epidermidis, viridans streptococci, Escherichia coli, Klebsiella Pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa.7

A number of tests are available to detect biofilm production by different bacteria. Methods include Tissue culture plate method, Tube method, Congo red agar, bioluminescent assay and light or fluorescent assay microscopic examination. In this study we screened isolates by tissue culture method to determine their ability to form biofilm.

Materials and Methods

This study was carried out from January 2019 to September 2020. During this period, 102 central venous lines were received in the microbiology laboratory. Positive cultures were obtained from 58 of these 102 samples. Peripheral vein sampling was done to rule out general bacteremia in all these patients to rule out seeding of the catheter through the blood. All cultured catheters were removed using sterile gloves after insertion site has been thoroughly cleaned with 2% povidine –iodine. After catheter was removed, the definition of catheter related infection was based on association of clinical signs (local or systemic)and significant catheter colonization. The distal 5cm of the catheter tip was cut with a sterile surgical scissors and put into separate container and then transported to microbiology laboratory in 2 hours for examination. To improve the detection of biofilm catheter vortexing was done. All catheter tips were cultured by the roll plate culture method directly onto 5% blood agar, Mac Conkey agar and chocolate agar. The plates were incubated at 37ºC aerobically for 24 hours. The number of organisms in the plates was evaluated quantitatively. A minimum of 15 colony forming units (CFU) in each plate was considered as positive catheter tip culture. Organisms were identified by standard microbiological procedures including various biochemical tests. All the isolates were subjected to antimicrobial susceptibility tests on Muller-Hinton agar by Kirby Bauer disc diffusion method based on CLSI guidelines.8

The isolates were then tested for in vitro production of biofilm using a microtitre plate assay. Biofilm-producing reference strains of A. baumannii (ATCC19606) and P. aeruginosa (ATCC 27853) were used as positive controls10 and non-biofilm forming reference strains of S. aureus (ATCC25923) and E. coli (ATCC25922) as negative controls.

Biofilm detection method

Tissue culture plate method (TCP)9

Organisms isolated from fresh agar plates were inoculated into 10 ml tryticase soy broth with 1% glucose (TSBglu). Broth was incubated at 370C for 18 hours. The broth culture was then diluted 1:100 with fresh medium. Individual wells of sterile 96 well flat bottom polystyrene tissue culture plates were filled with 200ul of the diluted cultures. Plates were incubated at 370C for 24 h. contents of each well were removed by gentle tapping. The wells were washed with 0.2 ml of phosphate buffer saline (ph7.2). Biofilm formed by bacteria adherent to the wells was fixed by 2% sodium acetate and stained by crystal violet (0.1%). Excess stain was removed by using deionized water and plates will be kept for drying. Optical density (OD) of stained adherent bacteria was determined with an ELISA reader at wavelength of 570nm. These OD values were considered as an index of bacterial adherence and biofilm formation.

Table 1

Classification of bacterial adherence by TCP method

Mean OD

Adherence

Biofilm Formation

<0.120    

Non

Non/weak

0.120-0.240    

Moderate

Moderate

>0.240

Strong

High

Ethical clearance

Ethical clearance was obtained from the Institute′s ethical clearance committee.

Results

The study was conducted in the clinical bacteriology laboratory, Department of Microbiology of Govt. Medical College, Srinagar and Kashmir. The central venous line received from patients admitted in SICU during January 2019 to December 2020 was processed for isolation and identification of bacterial pathogens according to the standard microbiological techniques.

Demographic characteristics of the study subjects showed that among 102 catheterized patients were isolated (age range 16-65 years) with males 60 (58.82%) and 42(41.1%) females respectively. There was no predilection of infection to any particular gender; patients of both genders were affected, males being marginally more than females. Majority (82%) of the patients were in the age group of 1-60 years, 21-40 yrs (36.27%); 1-20 yrs (27.45%) and 41-60 yrs (18.62%) respectively. A less number of patients were from elderly age groups >61 yrs (17%) as depicted in Table 1.

Table 2

Distribution of cases by age and gender (N= 102)

Age group (years)

Gender

Total No. of cases (n=102)

Male

Female

No.

%

1-20

15

13

28

27.45%

21-40

25

12

37

36.27%

41-60

10

9

19

18.62%

61-80

6

5

11

10.78%

>80

4

3

7

6.86%

Total

60 (58.82%)

42 (41.1%)

102

100

Out of 58 isolates, 36 (62.06%) were gram negative bacteria (GNB) and 22(37.93%) were gram positive (Figure 1).

Figure 1

Depicts the bacteriological profile of isolates

https://typeset-prod-media-server.s3.amazonaws.com/article_uploads/b2054102-df24-4d44-bf69-df63b19de08b/image/b2cda6af-0ccb-47ae-87b2-7c67d58c7d42-uimage.png

Table 3 depicts that 68% of the patients showed systemic signs of CRBI where as 32% of the patients showed local sins respectively.

Table 3

Distribution of catheter related bloodstream infection based on clinical signs (N=58)

Local signs

Systemic signs

Total

18(32%)

40(68%)

58(100%)

Table 4 shows that Staphylococcus aureus was most common isolate 16 (27.58%) followed by Klebsiella spp. 12 (20.68%), Acinetobacter 09 (15.5%), Coagulase negative Staphylococci 04 (6.8%) respectively.

Table 4

Shows different organisms isolated from central venous line (N=58).

Organisms

No.

%

Staphylococcus aureus

16

27.56

CONS

4

6.89

Enterococcus

1

1.72

Pneumococcus

1

1.72

Acinetobacter

9

15.51

Klebsiella

12

20.68

Pseudomonas

5

8.62

Enterobacter

5

8.62

Citrobacter

2

3.44

E.Coli

3

5.17

Isolates were screened for biofilm formation by TCP method. Out of 58 isolates, classification was done as Strong producer, Moderate producer, Weak/ Non-producer – 20.68%, 27.58%, 51.72% respectively based on their OD values as depicted in Table 5.

Table 5

Screening of the isolates for biofilm formation by tissue culture plate method in gram positive cocci

Organism

Weak

Moderate

Strong

Total

S. aureus

11

2

3

16

CONS

3

1

0

4

Enterococcus

0

1

0

1

Pneumococcus

1

0

0

1

Table 6

Shows total number of strong biofilm producers among gram positive cocci

Organism

Biofilm producers(No.)

Percentage

S. aureus

3

18.75

CONS

0

0

Enterococcus

0

0

Pneumococcus

0

0

Table 6 depicts screening of the isolates for biofilm formation by tissue culture plate method in gram negative bacilli.

Table 7

Screening of the isolates for biofilm formation by tissue culture plate method in gram negative bacilli

Organism

Weak

Moderate

Strong

Total

Acinetobacter

5

4

0

9

Pseudomonas

3

0

2

5

Klebsiella

5

3

4

12

E.coli

3

0

0

3

Enterobacter

2

3

0

5

Citrobacter

2

0

0

2

Table 8

Shows total number of strong biofilm producers among gram negative bacilli

Organism

Biofilm producers(No.)

Percentage

Acinetobacter

0

0

Pseudomonas

2

40

Klebsiella

4

33.3

E.coli

0

0

Enterobacter

0

0

Citrobacter

0

0

Antibiotic susceptibility patterns of various organisms isolated from central venous line. They showed increased resistance towards penicillin and beta-lactum group of antibiotics, increased sensitivity to linezolid and vancomycin among gram positive organisms. Among gram negative bacteria increased resistance was seen for cephalosporins and aminigylycosides and least resistance for colistin.

Antibiotic sensitivity pattern of different organisms isolated from central venous lines are:

Table 9

Shows antibiotic sensitivity pattern of biofilm producing gram positive bacteria

Organism

Sensitivity pattern

S. aureus

Vancomycin(100%)

Linezolid(100%)

Gentamicin(87.5%)

Cotrimoxazole(75%)

Clindamycin(62.5%)

Erythromycin (31.25%)

Ciprofloxacin (25%)

Cefoxitin(25%)

Penicillin (0%)

Table 10

Shows antibiotic sensitivity pattern of biofilmproducing Enterobacteriacae

Organism

Sensitivity pattern (%)

Klebsiella

Colistin(100%)

Imipenem(66.6%)

Ciprofloxacin (66.6%)

Cotrimoxazole(58.3%)

Cefepime(50%)

Gentamicin(50%)

PiperacillinTazobactum(41.6%)

Ceftazidime(33.3%)

Tobramycin(33.3%)

E. coli

Colistin(100%)

Imipenem(100%)

Amikacin(100%)

Ciprofloxacin (66%)

Ceftazidime-Tazobactum(66%)

Cefepime(33%)

Tobramycin(33%)

Enterobacter

Colistin (100%)

Imipenem(100%)

Amikacin(60%)

Ticarcillin-Clavulinic-acid (60%)

Ceftazidime(40%)

Cefepime(40%)

Piperacillin-Tazobactum(40%)

Ciprofloxacin(40%)

Table 11

Shows antibiotic sensitivity pattern of non fermenters

Organism

Sensitivity pattern

Acinetobacter

Colistin(100%)

Tigecycline(100%)

Minocycline(66.6%)

Gentamicin(44.4%)

Imipenem(33,3%)

Ciprofloxacin (33.3%)

Piperacillin-Tazobactum (11.1%)

Ceftriaxone(11%)

Cefepime(11%)

Pseudomonas

Colistin(100%)

Imipenem(80%)

Ciprofloxacin (80%)

Gentamicin(60%)

Piperacillin-Tazobactum(40%)

Cefepime(40%)

Ceftazidime-Clavulinic Acid(40%)

Discussion

Indwelling medical devices are frequently used in all health setting with critical care units of hospitals often using multiple medical devices for treatment and intervention in patient care.10 Intravascular catheters may be inserted for administration of fluids, blood products, medications, nutritional solutions, and hemodynamic monitoring. The organisms may originate from the skin of patients or healthcare workers, to which entry ports are exposed or other sources in the environment.7 Staphylococcus spp., Acinetobacter spp., Klebsiella spp Pseudomonas spp., and Enterobacter are the commonest causes of nosocomial infections. Colonization of indwelling medical devices with consequent biofilm production is a likely contributory factor to such infections. These microorganisms survive in the hospital environment despite unfavourable conditions such as desiccation, nutrient starvation, and antimicrobial treatment. It is hypothesized that the ability of these microbes to persist in these environments, as well as their virulence may be a result of their capacity to colonize medical devices.11

In the present study, a colonization rate of 53% was recorded in the test catheters. This was consistent with the study of Sadoyama et al (2006),12 who reported rates of colonization of (23-52%). The predominant contaminants were Gram-negative rods (62.06%). This is in agreement with Liniares et al(1985),13 Eyeret al(1990),14 Greogeret al (1993), 15 Tullu et al(1998),16 Sherertz et al (2000),17 Pelletier et al(2000),18 Hanna et al(2004)19 and Storti, et al(2005).20

The most prevalent Gram-negative rods isolated were identified as Klebsiella(20%), Acinetobacter(15%), Pseudomonas(8%), Enterobacter(8%). Alla et al.21 reported that the most prevalent Gram-negative rods isolated were identified as Acinetobacter baumannii (23%), followed by Pseudomonas aeruginosa (18%), Klebsiella pneumoniae (15%) and Enterobacter spp. (13%). However, different percentages of other Gram-negative isolates; including Klebsiella, Proteus, Pseudomonas, Stenotrophomonas, Enterobacter, Serratia as well as, Candida spp.; were reported by Shapiro et al (1982);22 Flynnet al(1987),23 Benezra D et al.(1988);24 Sherertz et al(1990),17 Baleva and Pena, (1997); 25 Seifert (1997), 26 Tulluet al(1998), 16 Mermel et al (2001), 27 Penaet al (2001),28 Cisneros and Rodriguez Bano, (2002),29 Krishnasami et al(2002)30 and Karlowsky et al(2004).31 This may be attributed to differences in culture techniques applied, catheterization periods, underlying diseases, types of catheters.

Gram-positive cocci, representing 37.9% of the isolates, were isolated, of these different authors (Darouiche et al 199932 and Paragioudaki et al 200433 reported much higher rates (60-67%).

The total number of positive slime producers, in this study, was 23(39.6%). The highest number of strong slime producer strains was observed in case of Pseudomonas (2 out of 5), Klebsiella pneumonia (4 out of 12) and Staphylococcus (3 out of 16) Farraget al (2002),34 Banin et al. (2006)35 Sherertz et al (2000)17 and Walencka et al(2006),36 also reported the ability of Pseudomonas for slime production.

The antibiogram of all bacterial isolates was performed using different antibiotics. The results obtained showed that the least resistance rates of Gram-negative bacteria were observed with colistin, tigicycline, imipenem and aminogycosides. All Gram-positive isolates were sensitive to linezolid, vancomycin followed by aminogycosides and cotrimoxazoles.

Source of Funding

None.

Conflict of Interest

None.

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Received : 12-05-2021

Accepted : 17-05-2021

Available online : 30-07-2021


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