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 ¤ Introduction
 ¤  Materials and Me...
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Table of Contents  
ORIGINAL ARTICLE
Year : 2011  |  Volume : 65  |  Issue : 9  |  Page : 387-392
 

Anti-biofilm and anti-adherence activity of Glm-U inhibitors


1 Department of Microbiology, Kasturba Medical College, Manipal University, Mangalore, India
2 Department of Community Medicine, Kasturba Medical College, Manipal University, Mangalore, India

Date of Web Publication16-Mar-2013

Correspondence Address:
Ethel Suman
Department of Microbiology, Kasturba Medical College, Manipal University, Mangalore - 575 001
India
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DOI: 10.4103/0019-5359.108954

PMID: 23508481

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 ¤ Abstract 

Background: Intravascular catheters and urinary catheters are an important source of hospital-acquired infections. Many microorganisms colonize indwelling catheters, including central venous catheters (CVCs) forming biofilms and cause infections that are difficult to treat. Although various methods have been employed to reduce biofilms, enzymes involved in bacterial cell wall synthesis could provide novel targets for the development of anti-biofilm agents. N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential enzyme in aminosugars metabolism and catalyzes the formation of uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc), an important precursor in the peptidoglycan and lipopolysaccharide biosynthesis of Gram-positive and Gram-negative bacteria. Previous study has been conducted on the anti-biofilm effect of GlmU inhibitors such as N-ethyl maleimide (NEM) and NEM analogs along with a cationic polypeptide protamine sulfate (PS), which enhanced its anti-biofilm activity. AIM: The present study aimed at finding the effect of sub-inhibitory concentrations of N-ethyl maleimide (NEM) and protamine sulfate (PS) on the biofilms produced by Pseudomonas aeruginosa and Enterococcus spp. isolated from cases of catheter-associated UTI as well as Klebsiella pneumoniae and Staphylococcus aureus isolated from cases of catheter-related bloodstream infections (CRBSI). Materials and Methods: In order to enhance the activity of NEM and to develop a broad-spectrum anti-microbial composition, NEM (50 μg/ml) was combined with protamine sulfate (50 μg/ml) and tested for anti-biofilm activity using a standard quantitative biofilm assay method. Results and Conclusion: It was observed that NEM had no effect on the biofilm produced by Pseudomonas aeruginosa as well as by Enterococcus spp. NEM also caused a significant decrease in biofilm production by Staphylococcus aureus while it had no effect on the biofilm produced by Klebsiella pneumoniae. There was a significant synergistic inhibitory effect on Staphylococcus aureus and Enterococcus spp., whereas Pseudomonas aeruginosa and Klebsiella pneumoniae remained unaffected. Combination of GlmU inhibitor-plus-protamine sulfate failed to significantly reduce bacterial adherence of Pseudomonas aeruginosa and Klebsiella pneumoniae to catheter and cannula pieces, respectively. We found that the GlmU inhibitor was mainly effective in preventing the adherence and biofilm formation by gram-positive organisms. The combination of NEM and protamine sulfate may, therefore, be tried as anti-infective coatings for medical devices such as catheters and cannulas, and thus help in overcoming microbial resistance in the current era of increasing device-associated hospital infections.


Keywords: Anti-biofilm, medical devices, N-Acetylglucosamine-1-phosphate uridyltransferase, N-ethyl maleimide, protamine sulfate


How to cite this article:
Suman E, D'souza SJ, Jacob P, Sushruth M R, Kotian M S. Anti-biofilm and anti-adherence activity of Glm-U inhibitors. Indian J Med Sci 2011;65:387-92

How to cite this URL:
Suman E, D'souza SJ, Jacob P, Sushruth M R, Kotian M S. Anti-biofilm and anti-adherence activity of Glm-U inhibitors. Indian J Med Sci [serial online] 2011 [cited 2014 Sep 18];65:387-92. Available from: http://www.indianjmedsci.org/text.asp?2011/65/9/387/108954



 ¤ Introduction Top


Intravenous and urinary catheters represent an essential part of the management of chronically and critically-ill patients. However, their use is often complicated by serious infections, mostly catheter-related bloodstream infections (CRBSIs) and urinary tract infections respectively, which are associated with increased morbidity, duration of hospitalization, and additional medical costs. [1] Many microorganisms colonize indwelling catheters, including central venous catheters (CVCs) forming biofilms. [2]

An area of great importance from a public health perspective is the role of biofilms in anti-microbial- drug resistance. Bacteria within biofilms are intrinsically more resistant to anti-microbial agents than planktonic cells because of the diminished rates of mass transport of anti-microbial molecules to the biofilm-associated cells [3] or because biofilm cells differ physiologically from planktonic cells. [4] The insensitivity of biofilm bacteria to antibiotics is a function of cell wall composition, surface structure, and phenotypic variation in enzymatic activity. [5]

It has also been suggested that the negatively charged exopolysaccharide is very effective in protecting bacterial cells from cationic antibiotics by restricting their permeation. [6] Various guidelines proposed to reduce the biofilms on catheters include use of intra-luminal anti-microbial lock solutions as well as minocycline-EDTA use as flush solution. [7] However, enzymes involved in bacterial cell wall synthesis could provide novel targets for the development of anti-biofilm agents. One of these enzymes is N-acetyl-D-Glucosamine-1-phosphate uridyl transferase (GlmU), which is involved in the biosynthesis of the activated nucleotide sugar UDP-GlcNAc, an essential precursor of peptidoglycans and lipopolysaccharides in Gram-positive and Gram-negative bacteria, respectively.

A previous study showed that N, N'(1,2 phenylene) dimaleimide (oPDM) an analogue of N-ethyl-maleimide (NEM) which is GlmU inhibitor when combined with a cationic polypeptide, protamine sulfate (PS) showed significant anti-biofilm activity. [8] The present study aimed at finding the effect of sub-inhibitory concentrations of N-ethyl maleimide (NEM) and protamine sulfate (PS) on the biofilms produced by Pseudomonas aeruginosa and Enterococcus spp. isolated from cases of catheter-associated UTI as well Klebsiella pneumoniae and Staphylococcus aureus isolated from cases of catheter-related bloodstream infections (CRBSI).


 ¤ Materials and Methods Top


Chemicals

The anti-biofilm compounds used in the study were protamine sulfate (PS and N-ethyl maleimide (NEM). Two different concentrations of anti-biofilm compounds were used for the study.

  • 1000 μg of each anti-biofilm compounds were dissolved in 1 ml of nutrient broth to get a solution of concentration 1000 μg/ml.
  • Anti-microbial activity of protamine sulfate plus N-ethyl maleimide combination was performed by dissolving anti-biofilm compounds in nutrient broth to get a solution of concentration 50 μg/ml.
Bacteria and culture conditions

The bacterial cultures used in this study included 10 strains of Pseudomonas aeruginosa and Enterococcus spp. isolated from catheter-associated urinary tract infection and 10 strains of Klebsiella pneumoniae and Staphylococcus aureus isolated from cases of CRBSI. Control strains included were Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, and Pseudomonas aeruginosa ATCC 27853. Antibiotic susceptibility test was performed for all the strains by modified Kirby Bauer disk diffusion method. [9]

The time required for killing the bacteria was determined by suspension test. [10] Broth macrodilution was used to determine the minimum inhibitory concentration and sub-inhibitory concentration of protamine sulfate and N-ethyl maleimide, respectively.

Biofilm assay

Biofilm assay was performed for all the organisms as per the method of O'Toole and Kolter. [11] The amount of biofilm produced was estimated spectrophotometrically, and the results were tabulated. The assay was repeated in the presence of sub-inhibitory concentration of protamine sulfate and N-ethyl maleimide individually as well as in combination.

Bacterial adhesion assay

Bacterial adhesion on catheter and cannula pieces was performed as follows. Uncoated Foley's catheter was sectioned with a scalpel to obtain 1 cm segments. Similarly, cannula was cut into 1 cm pieces. The inoculum was prepared by diluting overnight grown cultures of organisms to 1 in 10 in fresh BHI broth and incubated at 37°C for 2 h with shaking at 100 rpm. Catheter segments were immersed in artificial urine medium for 30 min, and cannula pieces were immersed in blood. After 30 min, the pieces were removed and placed in two sets of tubes containing 10 ml BHI broth. The first set of tubes were inoculated with 100 μl of bacterial inoculum (1 × 10 7 cells/ml), while the second set of tubes were inoculated with 100 μl of bacterial inoculum (1 × 10 7 cells/ml) as well as a combination of protamine sulfate and N-ethyl maleimide. The tubes were incubated in a waterbath for 3 h at 37°C with shaking at 100 rpm. After incubation, the pieces were washed three times in saline and transferred into a sterile tube containing 1 ml saline. Adherent bacteria were removed by vortexing the mixture for 2 mins. The cells were serially diluted in saline and plated onto nutrient agar plates. The plates were incubated at 37°C for 24 h, and the colonies were counted. Colony count of bacteria in the presence of the combination of anti-biofilm compounds was also recorded.

Statistical analysis

Statistical analysis was done using SPSS 15.0 and Kruskal Wallis test.


 ¤ Results Top


Anti-biofilm activity of GlmU inhibitor against catheter-associated uropathogen

In this study, we tested the anti-biofilm activity of GlmU inhibitor N-ethyl maleimide against Pseudomonas aeruginosa and Enterococcus spp. isolated from catheter-associated urinary tract infection. It was observed that NEM had no effect on the biofilm produced by Pseudomonas aeruginosa as well as by Enterococcus spp. [Figure 1].
Figure 1: Biofilm production in the presence of protamine sulfate and N-ethyl maleimide

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Anti-biofilm activity of GlmU inhibitor against pathogens isolated from CRBSI

NEM also caused a significant decrease in biofilm production by Staphylococcus aureus while it had no effect on the biofilm produced by Klebsiella pneumoniae [Figure 1].

Anti-biofilm activity of GlmU inhibitor-plus - protamine sulfate

In order to enhance the activity of NEM and to develop a broad-spectrum anti-microbial composition, NEM (50 μg/ml) was combined with protamine sulfate (50 μg/ml) and tested for anti-biofilm activity using a standard quantitative biofilm assay method. There was a significant synergistic inhibitory effect on Staphylococcus aureus and Enterococcus spp., whereas Pseudomonas aeruginosa and Klebsiella pneumoniae remained unaffected as shown in [Figure 1].

Anti-adhesion activity of GlmU inhibitor-plus - protamine sulfate

It was observed that the combination of GlmU inhibitor-plus-protamine sulfate failed to significantly reduce bacterial adherence of Pseudomonas aeruginosa and Klebsiella pneumoniae to catheter and cannula pieces, respectively, while it prevented the adherence the adherence of Staphylococcus aureus and Enterococcus spp.


 ¤ Discussion Top


The present study evaluated the anti-biofilm and anti-adherence activity of GlmU inhibitors against both gram-positive and gram-negative catheter-associated uropathogens and CRBSI-associated pathogens. GlmU inhibitors like NEM belong to a maleimide class of compounds that are quite reactive against sulfhydryl-containing enzymes and have poor specificity making them broad-spectrum anti-microbial compounds. [12] Protamine sulfate has been shown to enhance the activity of antibiotics. [13]

The fact that NEM was not effective against Pseudomonas aeruginosa and Klebsiella pneumoniae could be due to the higher MIC of maleimide for the organisms. When NEM was combined with protamine sulfate to increase the anti-biofilm activity, we observed a synergistic inhibitory activity, but it was seen only in case of the gram-positive organisms. This could be due to an alteration in membrane permeability and dilation of ionic channels by protamine sulfate, thereby facilitating the transport of NEM to the cytoplasm. This combination was also effective in inhibiting the adherence of the gram-positive organisms to catheter and cannula segments.

In summary, we found that the GlmU inhibitor was mainly effective in preventing the adherence and biofilm formation by gram-positive organisms. The combination of NEM and protamine sulfate may, therefore, be tried as anti-infective coatings for medical devices such as catheters and cannulas, and thus help in overcoming microbial resistance in the current era of increasing device-associated hospital infections.

 
 ¤ References Top

1.Stefanaia C, Fabrizio P, Nicola P. Clinical review: New technologies for prevention of intravascular catheter-related infections. Crit Care 2004;8:157-62.  Back to cited text no. 1
    
2.Elliott TS, Moss HA, Tebbs SE, Wilson IC, Bonser RS, Graham TR, et al. Novel approach to investigate a source of microbial contamination of central venous catheters. Eur J Clin Microbiol Infect Dis 1997;16:210-3.  Back to cited text no. 2
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3.Suci PA, Mittelman MW, Yu FP, Geesey GG. Investigation of ciprofloxacin penetration into Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother 1994;38:2125-33.  Back to cited text no. 3
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4.Evans DJ, Brown MR, Gilbert P. Susceptibility of Pseudomonas aeruginosa and Escherichia coli biofilms towards ciprofloxacin: Effect of specific growth rate. J Antimicrob Chemother 1991;27:177-84.  Back to cited text no. 4
    
5.Davies D. Understanding biofilm resistance to antimicrobial agents. Nat Rev drug Discov 2003;2:114-22.  Back to cited text no. 5
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6.Fux CA, Costerton JW, Stewart PS, Stoodley P. Survival strategies of infectious biofilms. Trends Microbiol 2005;13:34-40.  Back to cited text no. 6
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7.Raad I, Chatzinikolaou I, Chaiban G, Hanna H, Hachem R, Dvorak T, et al. In vitro and Ex vivo activities of minocycline and EDTA against microorganisms embedded in biofilm on catheter surfaces. Antimicrob Agents Chemother 2003;47:3580-5.  Back to cited text no. 7
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8.Burton E, Gauande PV, Yakandawala N. Antibio film activity of GlmU enzyme inhibitors against catheter associated uropathogens. Antimcrob Agents Chemother 2006;50:1835-40.  Back to cited text no. 8
    
9.Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing.15 th informational supplement:M100-S17 2007;26:75-76.  Back to cited text no. 9
    
10.Vizcaino-Alcaide MJ, Herruzo-Cabrera R. Fernandez-Acenero MJ. Comparison of the disinfectant efficacy of Perasafe and 2% glutaraldehyde in in vivo tests. J Hosp Infect 2003;53:124-8.  Back to cited text no. 10
    
11.O'Toole GA, Kolter R. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: A genetic analysis. Mol Microbiol 1998;28:449-61.  Back to cited text no. 11
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12.Filho VC, Pinhbiro T, Nunes RJ, Yunes RA. Antibacterial activity of N-phenylmaleimides, N-phenylsuccinimides and related compounds. Structure-activity relationships. Farmaco 1994;49:675-7.  Back to cited text no. 12
    
13.Sobho F, Khoury AE, Zamboni AC, Davidson D, Mittelman MW. Effects of ciprofloxacin and protamine sulfate combinations against catheter associated Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother 1995;39:1281.  Back to cited text no. 13
    


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