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BRIEF COMMUNICATION  
Year : 2013  |  Volume : 56  |  Issue : 3  |  Page : 276-278
Concomitant detection of biofilm and metallo-beta-lactamases production in gram-negative bacilli


1 Department of Microbiology, Government Medical College, Haldwani, Uttarakhand, India
2 Department of Biochemistry, All India Institute of Medical Sciences, Rishikesh, Uttarakhand, India

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Date of Web Publication24-Oct-2013
 

   Abstract 

Carbapenems are mainstay of treating serious multidrug resistant gram-negative biofilm-based infections. However, recent emergence of metallo-beta-lactamases (MbL) producing gram-negative bacilli in different parts of world may be related to gain of virulence factors associated with biofilm production. Objectives: To explore the association of MbL and biofilm production in various gram-negative bacilli. Materials and Methods: In this study, 110 non-repetitive ceftazidime resistant gram-negative bacilli were evaluated for biofilm and MβL production. Biofilm forming ability of isolates obtained from various specimens was tested by the tube method. Disks of ceftazidime (30 μg) and ceftazidime with ethylenediaminetetraacetic acid (30 μg + 750 μg, prepared in house) for MβL detection were used. Chi-square test was used to study the association between biofilm and MβL production. P value <0.05 was considered significant. Results: 88 (80%) bacilli had shown biofilm producing ability. The association of biofilm and MβL was significant in cases of non-fermenters as compared to enterobacteriaceae members. Conclusion: The particular combination of virulence factors (biofilm and MβL) in bacteria may be a species specific effect which needs to be investigated at molecular level in detail. This may help in designing newer therapies based on interference with biofilm formation and thus countering clinical episodes of antibiotic resistance.

Keywords: Biofilm, carbapenems, metallo-beta-lactamases

How to cite this article:
Singhai M, Rawat V, Goyal R. Concomitant detection of biofilm and metallo-beta-lactamases production in gram-negative bacilli . Indian J Pathol Microbiol 2013;56:276-8

How to cite this URL:
Singhai M, Rawat V, Goyal R. Concomitant detection of biofilm and metallo-beta-lactamases production in gram-negative bacilli . Indian J Pathol Microbiol [serial online] 2013 [cited 2019 Nov 18];56:276-8. Available from: http://www.ijpmonline.org/text.asp?2013/56/3/276/120394



   Introduction Top


Biofilm production is an inherent ability of bacteria which can lead to enhanced virulence due to gain of some additional phenotypic characters such as drug resistance. [1] Biofilms are formed after bacterial adherence to the surface and cell-wall-associated markers play an important role in this, therefore cell-wall inhibiting drug should be the drug of choice in such infections. [2] Carbapenems are cell-wall inhibiting agents and are mainstay of treating serious multidrug resistant gram-negative biofilm-based infections. However, recent outbreak of carbapenem resistant gram-negative bacilli such as Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, due to metallo-beta-lactamases (MβL) in different parts of world limits the choice of an appropriate and safe therapy. [2],[3],[4],[5] The evaluation of phenotypic resistance mechanism associated with biofilm mode of bacteria is essential to ensure proper antibiotic selection. As presence of MβL enzymes in gram-negative bacilli has been reported previously in our region, we explored the association of MβL and biofilm production in various gram-negative bacilli in this study. [3]


   Materials and Methods Top


A prospective study was carried out on 110 non-repetitive gram-negative bacilli resistant to ceftazidime on routine susceptibility testing obtained from hospitalized patients. The study was approved by institutional ethics committee. Minimum inhibitory concentration of isolates for meropenem was also determined by agar dilution method. The isolates were identified as per standard conventional methods and tested for in-vitro biofilm production and MβL production [Figure 1].
Figure 1: In vitro biofi lm production (left a and b) and metallo beta lactamase production (right)

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Detection of biofilms

Biofilm forming ability of isolates obtained from various specimens was tested by the tube method as done previously. [2],[6] Briefly 0.5 ml of suspension (0.5 McFarland of saline washed 24 h culture isolate) was inoculated into a polystyrene tube containing 4.5 ml of brain heart infusion broth and incubated at 37°C for 48 h without agitation. The culture broths from the tube were then aspirated, washed twice with distilled water and stained with crystal violet.

The biofilm forming isolates in tubes were scored as negative, weak, moderate or strong. Strong biofilm producer Staphylococcus epidermidis ATCC 35984 and non-biofilm producer Candida albicans ATCC 10231 were used as a positive and negative control respectively.

MβL detection

The gram-negative bacilli leading to resistance for ceftazidime on routine screening were confirmed for presence of MβL enzymes. Briefly, Muller-Hinton agar plates were prepared and inoculated with standardized inoculums to form a lawn culture. The combined disc methods were used to confirm above resistance mechanisms as described previously. [3] Disks of ceftazidime (30 μg) and ceftazidime with ethylenediaminetetraacetic acid, (EDTA) (30 μg + 750 μg, prepared in house) for MβL detection were used. The increase in inhibition zone with ceftazidime and EDTA disc ≥7 mm than the ceftazidime disc alone was considered as MβL positive.

Statistical analysis

The data were analyzed using stat-plus software. Chi-square test was used to study the association between biofilm and MβL production. P value <0.05 was considered significant.


   Results Top


A total of 110 non-repetitive ceftazidime resistant gram-negative bacilli were studied (66 enterobacteriaceae and 44 non-fermenters). 53 (49%) gram-negative bacterial isolates were also resistant to meropenem (MIC ≥8 μg/ml). 88 (80%) gram-negative bacilli had shown moderate to strong biofilm producing ability. 49 (44.5%) isolates had shown presence of MβL enzymes by disc combination method. Biofilm production and concomitant presence of MβL in different bacterial isolates are shown in [Table 1]. Biofilm production and presence of MβL enzymes was significantly correlated in non-fermenting gram-negative bacilli in our study. However, association of MβL and biofilm production among members of enterobacteriaceae was not found significant.
Table 1: Biofilm production and concomitant presence of mβl in gram negative bacilli

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   Discussion Top


Bacteria in biofilms might develop a biofilm-specific antibiotic resistant phenotype. [1] This may be attributed to particular resistance mechanism at work within a single community. In our previous study, we found many biofilm producing gram-negative isolates were extended spectrum beta lactamases (ESβLs) producers and a majority harbored particularly blaCTX-M, among the ESβLs genotypes. [7] The biofilm mode of bacterial growth and its importance vis-ΰ-vis β-lactam antibiotics insusceptibility due to presence of MβL enzymes was emphasized in this study. The rapid upsurge in number of MβL producing pathogens in various parts of world and that too residing in biofilm form is a problem of global concern. [3],[5],[8]

Biofilm production has been reported in strains of Pseudomonas spp and Acinetobacter spp and most infections are difficult to eradicate because they have a tendency to produce β-lactamases. [9],[10],[11] The association of biofilm and MβL was significant in cases of non-fermenters as compared to others. This study also signifies the presence of MβL producing phenotypes with ability to form biofilms among members of enterobacteriaceae, which although are rare but emerging pathogens and may be difficult to treat. [12] Therefore, we hypothesize that this particular combination of virulence factors (biofilm production and MβL) in bacteria is a species specific effect which needs to be investigated at genetic level in future. The concurrent detection of biofilm production and various resistance mechanisms among beta-lactam resistant gram-negative bacilli may help in designing newer therapies based on interference with biofilm formation and thus, countering clinical episodes of antibiotic resistance.

 
   References Top

1.Mah TF, O'Toole GA. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol 2001;9:34-9.  Back to cited text no. 1
    
2.Singhai M, Malik A, Shahid M, Malik MA, Goyal R. Characterization of fungal biofilm-based catheter-related sepsis. Chron Young Sci 2012;3:48-52.  Back to cited text no. 2
  Medknow Journal  
3.Rawat V. Study of mettalo-β-talactamses production in noscomial nil fermenter gram negative bacilli in a tertiary care centre. Int J App Basic Med Res 2011;1:139-40.  Back to cited text no. 3
    
4.Deshpande P, Rodrigues C, Shetty A, Kapadia F, Hedge A, Soman R. New Delhi Metallo-beta lactamase (NDM-1) in Enterobacteriaceae: Treatment options with carbapenems compromised. J Assoc Physicians India 2010;58:147-9.  Back to cited text no. 4
    
5.Lagatolla C, Tonin EA, Monti-Bragadin C, Dolzani L, Gombac F, Bearzi C, et al. Endemic carbapenem-resistant Pseudomonas aeruginosa with acquired metallo-beta-lactamase determinants in European Hospital. Emerg Infect Dis 2004;10:535-8.  Back to cited text no. 5
    
6.Singhai M, Malik A, Shahid M, Malik A, Rawat V. Colonization of peripheral intravascular catheters with biofilm producing microbes: Evaluation of risk factors. Niger Med J 2012;53:37-41.  Back to cited text no. 6
[PUBMED]  Medknow Journal  
7.Singhai M, Malik A, Shahid M, Malik MA, Goyal R. A study on device-related infections with special reference to biofilm production and antibiotic resistance. J Glob Infect Dis 2012;4:193-8.  Back to cited text no. 7
    
8.Munoz-Price LS, Weinstein RA. Acinetobacter infection. N Engl J Med 2008;358:1271-81.  Back to cited text no. 8
    
9.Chakraborty D, Basu S, Chatterjee P, Dey SK, Das S. Concurrent determination of collagenase and biofilm formation activities in metallo-beta-lactamase producing Pseudomonas aeruginosa. Int J Microbiol Res 2011;2:208-12.  Back to cited text no. 9
    
10.Rao RS, Karthika RU, Singh SP, Shashikala P, Kanungo R, Jayachandran S, et al. Correlation between biofilm production and multiple drug resistance in imipenem resistant clinical isolates of Acinetobacter baumannii. Indian J Med Microbiol 2008;26:333-7.  Back to cited text no. 10
[PUBMED]  Medknow Journal  
11.Perez LR, Antunes AL, Freitas AL, Barth AL. When the resistance gets clingy: Pseudomonas aeruginosa harboring metallo-β-lactamase gene shows high ability to produce biofilm. Eur J Clin Microbiol Infect Dis 2012;31:711-4.  Back to cited text no. 11
    
12.Poulou A, Spanakis N, Pournaras S, Pitiriga V, Ranellou K, Markou F, et al. Recurrent healthcare-associated community-onset infections due to Klebsiella pneumoniae producing VIM-1 metallo-beta-lactamase. J Antimicrob Chemother 2010;65:2538-42.  Back to cited text no. 12
    

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Correspondence Address:
Monil Singhai
Department of Microbiology, Government Medical College, Haldwani, Uttarakhand
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.120394

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