|Year : 2022 | Volume
| Issue : 1 | Page : 13-17
Effect Biofilm Formation in Pseudomonas aeruginosa Resistance To Antibiotic
Hanan Hameed Shatti1, Wisam M Al-Saeed2, Mohammed I Nader3
1 Department of Laboratory, Iraqi Ministry of Health, Baghdad, Iraq
2 Department of Microbiology, College of Medicine, Mustansiriyah University, Baghdad, Iraq
3 Institute Genetic Engineering and Biotechnology for Post Graduated Studies, University of Baghdad, Baghdad, Iraq
|Date of Submission||28-Jul-2021|
|Date of Decision||21-Aug-2021|
|Date of Acceptance||28-Aug-2021|
|Date of Web Publication||30-Jun-2022|
Miss. Hanan Hameed Shatti
Department of Laboratory, Iraqi Ministry of Health, Baghdad
Source of Support: None, Conflict of Interest: None
Objective: Due Pseudomonas aeruginosa is the most common type of bacteria that causes hospital-acquired infections because it has multiple resistance mechanisms to antibiotics such as biofilm formation. It is important to understand to investigate biofilm production in multidrug resistant clinical isolates of P. aeruginosa from different sites of infection. Materials and Methods: This work was done on 114 patients were collected from different specimens From hospitals Baghdad. The collected samples were cultured on different media (blood agar, MacConkey agar, nutrient agar, and Cetrimide agar) for isolation of P. aeruginosa bacteria as well as isolates from all patient were tested for antimicrobial susceptibility using disk diffusion method ,in vitro formation of biofilm in microtiter plates containing Muller –Hinton broth. Results: Forty specimens (35.1%) were observed to have bacterial growth (positive samples) for P. aeruginos, the results of P. aeruginosa isolates were tested for antibiotic susceptibility showed that among forty positive results, the most isolates were potentially resistant to different antibiotics presented that the isolates resistant to piperacillin (34, 85%), gentamicin (26, 65%), tobramycin (30, 82.5%), amikacin (27, 67.5%), cefepime (25, 62.5%), meropenem (31, 77.5%), carbenicillin (31, 77.5%), ceftriaxone (30, 75%), ciprofloxacin (27, 67.5%), imipenem (22, 55%), ceftazidime 26 (65%), and norfloxacin 24 (60%). The results showed the ability of P. aeruginosa isolates to produce biofilm were 40 (100%) has the ability to produce biofilm, these were the result divided in to strong 19 (47.5%), moderate 12 (30%), and weak 9 (22.5%). Conclusion: There were 40 isolates biofilm producers in divers levels of biofilm strength.
Keywords: Antimicrobiogram biofilm, Pseudomonas aeruginosa
|How to cite this article:|
Shatti HH, Al-Saeed WM, Nader MI. Effect Biofilm Formation in Pseudomonas aeruginosa Resistance To Antibiotic. Mustansiriya Med J 2022;21:13-7
|How to cite this URL:|
Shatti HH, Al-Saeed WM, Nader MI. Effect Biofilm Formation in Pseudomonas aeruginosa Resistance To Antibiotic. Mustansiriya Med J [serial online] 2022 [cited 2022 Dec 2];21:13-7. Available from: https://www.mmjonweb.org/text.asp?2022/21/1/13/349302
| Introduction|| |
Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium belonging to the family Pseudomonadaceae that is able to survive in a wide range of environments. The opportunistic pathogen P. aeruginosa can infect a variety of organisms, including plants, animals, and most importantly, humans, in particular, immunocompromised, burn-wound patients and individuals suffering from cystic fibrosis. P. aeruginosa has many virulence factors such as pili, cilia, large polysaccharide (LPS), extracellular enzymes, exotoxins, and flagella. These bacteria contain a single polar flagellum, which is essential for movement, chemotaxis, and adhesion. One of the major virulence factors for this pathogen is its ability to form biofilms. This biological development protects the pathogen from host immunity and contributes to its antimicrobial resistance. It is estimated that about 80% of infectious diseases are due to biofilm formation. Biofilm-forming ability and antimicrobial resistance of this pathogen lead to many persistent and chronic bacterial infections. A biofilm can be described as a microbially derived sessile community characterized by cells. These cells are irreversibly attached to a surface or interface or to each other, are inserted in a matrix of extracellular polymeric substances (EPSs) that they have produced, and exhibit an altered phenotype in terms of growth rate and gene transcription Biofilm formation is an important virulence factor increase the pathogenicity infectious of P. aeruginosa. The infection instigated by biofilm former P. aeruginosa can proceed for chronic form due to traceability of antibiotic effects. The outstanding of biofilms to antibiotics is multifactorial including physic-physiological and genetic determinants.
| Materials and Methods|| |
Through the period from November 2020 to February 2021, 114 isolates were collected from different specimens from different clinical sources from several hospitals in Baghdad governorate (Imam Kadhimin Medical City, Hospital, Al-Yarmouk Teaching Hospital, Al-Mahmoudia General Hospital) from both gender with ages ranged from 5 years to 75 years.
Bacterial isolation and identification
The isolates were identified based on the colony morphology on different culture media MacConkey agar, Nutrient agar, Blood agar, and Cetrimide agar, incubated for 24 h at 37°C. This isolates may belong to Paeruginosa growth on Cetrimide agar, in conjunction with traditional biochemical tests comprised gram staining, oxidase, catalase, oxidation/fermentation glucose, and growth and nongrowth at 42°C and 4°C, respectively, were performed to characterize P. aeruginosa colony morphologically, cell microscopically, and metabolically by ByAnalytical Profile Index (API) 20E Kit Biomerieux® company (France and USA), microtubules enzymatically tests to support strains identification, and the isolated P.aeruginosa which stored in brain heart infusion (BHI) broth contains 30% glycerol at refrigerator temperature and −20°C, respectively, for the next experiments.
The disk diffusion susceptibility method for antibiogram testing (Kirby–Bauer method) using Muller–Hinton agar plates was performed according to the Clinical and Laboratory Standards Institute. Antibiotic discs (Bioanalyse – Turkey) used in this study included tobramycin, ciprofloxacin, imipenem, meropenem–cefepime, ceftriaxone, gentamicin, piperacillin, ceftazidime, carbenicillin, amikacin, and norfloxacin. After an overnight incubation at 37°C, zones of inhibition were measured and compared to the CLSI guidelines.
Quantitative biofilm formation assay
Quantification of biofilm formation by P. aeruginosa was assessed, as described by the previous study. In brief:
- All isolates were grown overnight in BHI broth at 37°C. Thereafter, it was adjusted to McFarland standard No. (0.5)
- Each isolate were transferred to Muller–Hinton broth containing 1% glucose and mixed well via pipetting. Thereafter, it was adjusted to McFarland standard No. (0.5)
- A volume (200 μl) for each isolate culture was added to duplicate of sterile 96-well U-shaped-bottom polystyrene microplates
- The plates were covered with their lids and incubated under aerobic conditions at 37°C for 24 h
- After the incubation period, the planktonic cells were rinsed twice with deionized water to remove the unattached bacteria and then shaken out the excess water by tapping the plate on paper towels (filter paper), dried, and fixed at 65°C for 1 h
- The adhering bacterial cells in each well were fixed with 200 μl of absolute methanol with a concentration of 99% for 20 min at room temperature
- The adhering cells were stained by adding 200 μl of 0.1% crystal violet to each well for 15 min
- Once the staining reaction has completed, the excess stain was removed and left to dry., then glacial acetic acid was added at a concentration of 33% and at a rate of 160
- Then, the optical density was read by enzyme-linked immunosorbent assay reader (ELISA reader) at a wavelength of 630 nm to determine the efficiency of the isolates in biofilm production and compared with the equations mentioned in [Table 1].
|Table 1: Classification of bacterial adherence by microtiter plate method|
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| Results|| |
Isolation and identification of P. aeruginosa by traditional methods
A total of 40 (35%) isolates developed a growth on cetrimide agar blue-greenish color, mucoid colony, smooth in shape, and have a fruity odor showed in [Figure 1], pale colonies on MacConkey agar. Moreover, they were oxidase and catalase positive. Hence these isolates were primarily identification as p aeruginosa, the results of media growth and biochemical tests are listed in [Table 2].
|Table 2: Results of biochemical test for identification Pseudomonas aeruginosa isolates from different clinical samples|
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|Figure 1: Colonies of Pseudomonas aeruginosa on cetrimide agar plate at 37°C for 24 h|
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For more accurate detection, by using the strip of API 20E metabolically and enzymatically assay, in [Figure 2].
The API 20E results revealed that 40 isolates tested positive for belonging to the P.aeruginosa species.
Antimicrobiogram susceptibility test
Forty of P. aeruginosa were subjected to an antibiogram examination according to the Clinical and Laboratory Standards Institute to determine possible resistance to 12 antibiotics items from 5 separate groups The results were interpreted according to the recommendation of CLSI (2019). P. aeruginosa exhibited diversity in resistance phenomena, the isolates were the highest rate of resistance against to piperacillin (34, 85%), meropenem (31, 77.5%), carbenicillin (31, 77.5%), tobramycin (30, 75%), ceftriaxone (30, 75%), ciprofloxacin (27, 67.5%), gentamicin (26, 65%), and ceftazidime (26, 65%), then percentage resistance of isolates began gradually to decline with cefepime (25, 62.5%), norfloxacin (24, 60%), and imipenem with percentages as 22 (55%). These isolates showed different susceptibility toward these antibiotics, as shown in [Table 3].
The capacity of all (40) P. aeruginosa isolates to generate biofilm was determined in this study using standard microtiter plates and reading in an automated ELISA reader, using a 630 nm wavelength, the absorbance of stained biofilms was measured. The results revealed that 40 (100%) isolates were adhered and capable of forming a positive biofilm. These positive results were divided into there were 47.5% strong biofilm formation, 30.0% moderate and 22.5% weak. The weak biofilm formation is shown in [Figure 3].
| Discussion|| |
P. aeruginosa is a major pathogen in hospitalized patients, causing morbidity and mortality due to its multiple resistance mechanisms. The findings of this study revealed that 40 (35.1%) of the total 114 samples isolated from patients were positively identified as P. aeruginosa. The results were similar to this study (37.9%) from hospitalized burn patients in Karbala. Moreover, other results that showed 55 (37.93%) of the total 145 sample isolates from burn patients were positively diagnosis as P. aeruginosa and this may be attributed to the highest prevalence of this genus of bacteria P. aeruginosa in the contamination and nature. P. aeruginosa is distinguishable from other types of bacteria by its capability for growth at 42°C and formation of blue–greenish color pigments associated with P.aeruginosa on selective medium cetrimide agar and this result agreement with the previous study. Antibiotic sensitivity testing is an essential tool for treatment. P. aeruginosa isolates showed high prevalence of resistance against, β-Lactamase inhibitors class (piperacillin, carbenicillin) 85%, 77.5% respectively. Due to P. aeruginosa can produce β-lactamase enzymes is the most common and important mechanism of resistance in Gram-negative bacteria. β-lactamase diffuses through or directly traverse porin channels in the outer membrane of Gram-negative bacterial cell walls. P. aeruginosa either decreased porin production or increased efflux and reduced permeability. Hence, the result showed resistance to meropenem (31, 77.5%), tobramycin (30, 75%), ceftriaxone (30, 75%), ciprofloxacin (27, 67.5%), gentamicin (26, 65%), and ceftazidime (26, 65%), then percentage resistance of isolates began gradually to decline with cefepime (25, (62.5%), norfloxacin (24, 60%), and imipenem with percentages as 22 (55%). The study showed that the multidrug resistant (MDR) which is resistant to three classes of antimicrobials increased among these organisms making it difficult to choose appropriate suitable antimicrobial therapy, and the increased resistance of P. aeruginosa to numerous antibiotics, as a result of excessive antibiotic administration, is now leading to the accumulation of antibiotic resistance and cross-resistance between antibiotics and the appearance of MDR forms of P. aeruginosa. The method microtiter of the plate is very important in the early study due to determining the membrane configuration and classification of production to high, medium, and weak for biofilm formation are very important because of the factors of the survival and protection of bacteria in addition to its role in the adhesion on the surface, especially in the hospital environment and it causes acquired disease so. Regarding the biofilm, the result in this study showed that 100% of isolates produced biofilm formation, there were 47.5% strong biofilm formation, 30.0% moderate, and 22.5% weak biofilm formation. This result was compatible with other studies that showed 32 (91.4%) of P. aeruginosa isolates were biofilm producers and 25.7%, 40%, and 25.7% of isolates were strong, moderate, and weak biofilm producers, respectively. In Iranian investigations found that all isolates from burn patients (92.4%). In India, isolates forming the biofilm were resistant to all antibiotics, and this resistance increased in these isolates by 10–100 times compared to the isolates that did not form the biofilm. Thus, the formation of the biofilm is one of the most important virulence factors important in the treatment of infections caused by bacteria. In addition, the grade and strength of biofilm development are determined by the location and form of infection, the length of bacterial invasion, host immune activity, the composition of EPS, and environmental adaptation. The high percentages in the current study of bacterial production of the biofilm may explain the high rate of bacterial resistance to all antibiotics, as the biofilm plays a major role in pathogenicity because it forms the mucous layer and the host's proteins, which provides the appropriate place for the growth of bacteria and their resistance to treatment. The discrepancy in results between different studies may be attributed to many factors such as the different countries from which the samples were collected, the number and the type of clinical specimens from which the isolates were obtained, and also the differences in isolates capability to form biofilm.
| Conclusion|| |
P. aeruginosa isolates differ in their ability to produce biofilm formation, bacteria that produce biofilm more resistant to the antibiotic.
- Studying the other mechanisms of antibiotic resistance
- Determine genes related to the formation of the biofilm.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Pachori P, Gothalwal R, Gandhi P. Emergence of antibiotic resistance Pseudomonas aeruginosa
in intensive care unit; a critical review. Genes Dis 2019;6:109-19.
Garcia M, Morello E, Garnier J, Barrault C, Garnier M, Burucoa C, et al. Pseudomonas aeruginosa
flagellum is critical for invasion, cutaneous persistence and induction of inflammatory response of skin epidermis. Virulence 2018;9:1163-75.
Elmanama AA, Al-Sheboul S, Abu-Dan RI. Antimicrobial resistance and biofilm formation of Pseudomonas aeruginosa. Int Arab J Antimicrob Agents 2020;10:3.
Flemming HC, Wingender J, Szewzyk U, Steinberg P, Rice SA, Kjelleberg S. Biofilms: An emergent form of bacterial life. Nat Rev Microbiol 2016;14:563-75.
Issa MA, Ghaima KK, Saleh MB, Nader MI. Antibiogram study and prevalence of pslá gene among biofilm Pseudomonas aeruginosa
producers isolated from some clinical specimens in thi_qar province. Pak J Biotechnol 2018;15:681-8.
Ciofu O, Tolker-Nielsen T. Tolerance and resistance of Pseudomonas aeruginosa
biofilms to antimicrobial agents-how P. aeruginosa
can escape antibiotics. Front Microbiol 2019;10:913.
Gillespie S, Hawkey PM. Principles and Practice of Clinical Bacteriology. John Wiley and Sons; 2006.
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing, M100-S29. 29th
ed. Clinical and Laboratory Standards Institute; 2019.
Al-Ouqaili MT, Al-Kubaisy SH. Crystalline biofilm produced by Proteus mirabilis
: An overview on their formation assays and antimicrobial interaction. Al Anbar J Med 2008;6:33-42.
Al-Daraghi WA, Al-Badrwi MS. Molecular detection for nosocomial Pseudomonas aeruginosa
and its relationship with multidrug resistance, isolated from hospitals environment. Med Leg Updat 2020;20:631-6.
Shilba AA, Al-Azzawi RH, Al-Awadi SJ. Dissemination of carbapenem resistant Pseudomonas aeruginosa
among burn patients in Karbala Province\Iraq. Iraqi J Sci 2015;56:1850-7.
Ismail ST, Altaai MI. Study ndvB gene expression in Pseudomonas aeruginosa
producing biofilm. Med Leg Updat 2021;21:961-5.
Al-Shamaa NF, Abu-Risha RA, Al-Faham MA. Virulence genes profile of Pseudomonas aeruginosa local isolates from burns and wounds. Iraqi J Biotechnol 2016;15:31-9.
Ammeter D, Idowu T, Zhanel GG, Schweizer F. Development of a nebramine-cyclam conjugate as an antibacterial adjuvant to potentiate β-lactam antibiotics against multidrug-resistant P.aeruginosa
. J Antibiot (Tokyo) 2019;72:816-26.
Yayan J, Ghebremedhin B, Rasche K. Antibiotic resistance of Pseudomonas aeruginosa
in pneumonia at a single university hospital center in Germany over a 10-year period. PLoS One 2015;10:e0139836.
Pedersen SS, Høiby N, Espersen F, Koch C. Role of alginate in infection with mucoid Pseudomonas aeruginosa
in cystic fibrosis. Thorax 1992;47:6-13.
El-Khashaab TH, Erfan DM, Kamal A, El-Moussely LM, Ismail DK. Pseudomonas aeruginosa
biofilm formation and quorum sensing lasR gene in patients with wound infection. Egypt J Med Microbiol 2016;38:1-8.
Ghanbarzadeh Corehtash Z, Khorshidi A, Firoozeh F, Akbari H, Mahmoudi Aznaveh A. Biofilm formation and virulence factors among Pseudomonas aeruginosa
isolated from burn patients. Jundishapur J Microbiol 2015;8:e22345.
Hall-Stoodley L, Stoodley P. Evolving concepts in biofilm infections. Cell Microbiol 2009;11:1034-43.
Algburi A, Comito N, Kashtanov D, Dicks LM, Chikindas ML. Control of biofilm formation: Antibiotics and beyond. Appl Environ Microbiol 2017;83:e02508-16.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]