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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 16  |  Issue : 2  |  Page : 357-362

Increased expression of genes involved in biofilm formation in a multidrug-resistant environmental Pseudomonas aeruginosa isolate


Nitte (Deemed to be University), Nitte University Centre for Science Education and Research, Paneer Campus, Deralakatte, Mangaluru - 575018, Karnataka, India

Date of Submission29-Jul-2020
Date of Decision21-Nov-2020
Date of Acceptance01-Dec-2020
Date of Web Publication18-Oct-2021

Correspondence Address:
Dr. Ramya Premanath
Nitte (Deemed to be University), Nitte University Centre for Science Education and Research, Paneer Campus, Deralakatte, Mangalore - 575 018, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdmimsu.jdmimsu_286_20

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  Abstract 


Introduction: Pseudomonas aeruginosa is an important opportunistic pathogen associated with majority of the hospital-acquired infections in immunocompromised patients, worldwide. Biofilms formed by this bacterium increase its resistance toward antibiotics, which in turn increase morbidity and mortality. As environmental isolates of P. aeruginosa are also capable of producing virulence-associated traits, they can be considered as potential pathogens. Materials and Methods: This study was undertaken to compare the expression of some of the biofilm genes such as algD, pslA, pslB, pelA, and pelD in environmental and clinical isolates of P. aeruginosa isolated from Coastal Karnataka. The expression of biofilm-forming genes in strong biofilm-forming P. aeruginosa isolated from sputum (HS26), wound swab (HW20), and environment (EN4) were quantified at different time points of incubation (18, 25, 48, and 72 h). In addition, the sensitivity of the selected isolates to commonly used antibiotics (amikacin, aztreonam, carbenicillin, ciprofloxacin, cefotaxime, imipenem, and gentamicin) was investigated. Results: The current investigation revealed the presence of a multidrug-resistant environmental isolate with higher expression of the biofilm genes as compared to the clinical ones. In the environmental isolate, the relative expression of pslA, pslB, pelD, and pelA gene was increased by 11, 10, 10, and 9 folds, respectively, at 48 h. Conclusion: The study suggests the possibility of such multidrug-resistant biofilm-forming environmental isolates of P. aeruginosa getting transferred to the hospital setting and causing life-threatening biofilm-associated infections.

Keywords: Biofilm, environmental isolate, gene expression, Pseudomonas aeruginosa


How to cite this article:
Alva PP, Sundar S, D'Souza C, Premanath R. Increased expression of genes involved in biofilm formation in a multidrug-resistant environmental Pseudomonas aeruginosa isolate. J Datta Meghe Inst Med Sci Univ 2021;16:357-62

How to cite this URL:
Alva PP, Sundar S, D'Souza C, Premanath R. Increased expression of genes involved in biofilm formation in a multidrug-resistant environmental Pseudomonas aeruginosa isolate. J Datta Meghe Inst Med Sci Univ [serial online] 2021 [cited 2021 Dec 9];16:357-62. Available from: http://www.journaldmims.com/text.asp?2021/16/2/357/328461




  Introduction Top


Pseudomonas aeruginosa is one of the leading multidrug-resistant pathogens causing opportunistic nosocomial infection. Resistance in P. aeruginosa poses a serious therapeutic challenge, which significantly increases morbidity, mortality, and overall cost of treating the infection. It is one of the leading causes of morbidity and mortality among compromised individuals. P. aeruginosa survives in diverse ecological niche. It can utilize a wide variety of carbon sources for energy, and due to this metabolic versatility, it exists in environments such as soil, water, plants, animals, humans, and many of the artificial environments throughout the world. Its survival in a diverse ecological niche is aided by its ability to form polysaccharide-encased, surface-attached communities called as biofilms. Biofilms are symbolically called the “City of microbes.”[1] They are microbial communities encased in extracellular polymeric substances that allow the organism to survive in hostile environments.[2] It represents sessile communities that are different morphologically and physiologically from free living ones. Biofilms formed by the microorganisms serve as their immediate environment and are of clinical and environmental relevance. Polysaccharides, proteins, nucleic acids, and lipids that make up the biofilms provide stability, bring about adhesion to surfaces, and form a network that interconnects and immobilizes biofilm cells.[3]

The stability of the biofilm structure in P. aeruginosa is determined by the production of three polysaccharides namely, Pel, Psl, and alginate. Alginate is a linear polymer of D-mannuronic acid and L-guluronic acid, responsible for structural stability, water, and nutrient retention and also protection. Psl is a pentasaccharide made up of D-glucose, D-mannose, and L-rhamnose. Pel polysaccharide is mainly composed of glucose-rich material.[4] Early stages of biofilm formation are marked by the involvement of Pel and Psl which forms a primary structure.[5] The resistance of this pathogen to antibiotics increases when growing as biofilms than when they are in a planktonic state. Formation of P. aeruginosa biofilms not only occurs in nature but also inside the host, leading to chronic lung infections in patients with cystic fibrosis, in patients with indwelling catheters, and in patients with other prosthetic materials.[6] las, rhl, and PQS systems have been recognized as the three quorum sensing systems in P. aeruginosa that propel the synthesis and perception of autoinducer signaling molecules, all of which are known to play roles in biofilm formation.[7] Bacteria in biofilms are extremely resistant to a large number of antimicrobial compounds, the mechanisms for which are multifactorial. Although studies have been carried out to demonstrate the biofilm formation in clinical isolates of P. aeruginosa from cystic fibrosis patients, hardly few studies have been conducted to evaluate their level of gene expression. Furthermore, there are limited studies on biofilm formation and its level of expression in environmental isolates. This prompted us to undertake the current investigation to examine the presence of few biofilm forming genes, their ability to form biofilm at different time intervals, as well as their expression.


  Materials and Methods Top


Revival of clinical and environmental isolates of Pseudomonas aeruginosa

A total of 90 P. aeruginosa isolates (sputum = 30, wound swab = 30, environment [soil and water] = 30) were revived from the glycerol stock (−80°C) maintained in our institute. The isolates were inoculated into Luria-Bertani broth and incubated at 37°C overnight. Isolates were reconfirmed as P. aeruginosa by plating on cetrimide agar. P. aeruginosa PAO1 was used as the reference strain.

Antibiotic susceptibility testing

P. aeruginosa isolates were checked for their antibiotic susceptibility by Kirby–Bauer disc diffusion method as described by CLSI guidelines (CLSI 2017). A loop full of overnight culture was used to inoculate 5 ml Luria-Bertani broth and incubated at 37°C with shaking until light-to-moderate turbidity was obtained. The turbidity was compared with that of standard 0.5 McFarland. The culture was then swabbed onto Muller–Hinton agar plates and antibiotic discs (amikacin [30 mcg], aztreonam [30 mcg], carbenicillin [100 mcg], ciprofloxacin [5 mcg], cefotaxime [30 mcg], imipenem [10 mcg], and gentamicin [10 mcg]) were placed aseptically. The plates were incubated at 37°C for 16–18 h and zone of clearance was measured and interpreted as susceptible, intermediate, or resistant.

Amplification of biofilm-associated genes

Bacterial crude DNA isolation was performed using the CTAB method, and the isolated DNA was preserved at −20°C until further use. The DNA obtained from clinical and environmental isolates was subjected to polymerase chain reaction (PCR) using primers specific for biofilm-forming genes, viz., algD, pslA, pslB, pelA, and pelD [Table 1]. All the primers were designed using Primer 3 input version 0.4.0 (Steve Lincoln, Mark Daly, and Eric S. Lander). PCR was carried for 30 cycles (Eppendorf, Germany) and the PCR product was analyzed by agarose gel electrophoresis (BioRad, USA).
Table 1: Name and sequence of primers used for identification of biofilm-associated genes

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Quantification of biofilm

Biofilm quantification was carried out according to the method of O'Toole and Kolter[8] with a minor modification. 100 μl of the diluted culture was taken in a microtiter plate and incubated for 24 h at 37°C. The adherent cells were washed thrice with phosphate-buffered saline of pH 7.4. 125 μl of 0.1% freshly prepared crystal violet was added to the dried pellet and incubated for 10 min. To the stained and washed pellet, 200 μl of 30% acetic acid was added and incubated for 15 min for stain solubilization. 100 μl was transferred to the fresh plate and optical density (OD) was measured at 600 nm in an ELISA reader (BioRad, USA). The biofilm formed by the isolates of P. aeruginosa was compared with the reference strain. The biofilm formers were grouped as nonbiofilm formers (OD600 ≤0.071), weak biofilm formers (OD600 0.071–0.142), moderate biofilm formers (OD600 0.142–0.284), and high biofilm formers (OD600 ≥0.284).[9]

Quantification of biofilm genes in Pseudomonas aeruginosa isolates

Isolates of P. aeruginosa having a potential for strong biofilm formation representing each of the sputum, wound swab, and environment were compared with the reference strain for the expression of biofilm-associated genes algD, pslA, pelA, pslB, and pelD. RNA extraction was performed by TRIzol method. Obtained RNA was further subjected to DNase treatment for the removal of contaminating DNA. Primer concentration and its efficiency were analyzed before real-time PCR. Different concentrations of primer (100, 200, and 300 nM) were used for the standardization. ΔΔct efficiencies of the primer were validated by amplifying serially diluted DNA. Amplification efficiencies of reference and target primers were indicated by slope of the graph being close to 0. cDNA synthesis was carried out using PrimeScript1st strand cDNA synthesis kit (Takara, Japan). SensiFAST SYBR kit (Proteogen Biosciences, India) was used to carry out the quantitative real-time PCR (qPCR). Melt curve analysis was performed at the end of 39 cycles to check for the presence of unique PCR reaction product. To detect the presence of contaminating DNA, a no-template control was included. Ribosomal rpsL gene was used as an internal control, and P. aeruginosa PAO1 was used as the calibrator. Analysis of relative gene expression was by the 2−ΔΔct method.[10] Data acquisition was performed by CFX96™ (BioRad) Manager at the end of each elongation step.

Statistical analysis

The data were subjected to one-sample t-test using SPSS, 16.0 software (IBM, USA) and data were considered to be statistically significant at P ≤ 0.05.


  Results Top


Antibiotic susceptibility testing

There was presence of multidrug-resistant strains of P. aeruginosa based on their antibiotic susceptibility profile [Figure 1]. Large number of sputum and wound swab isolates was resistant to cefotaxime. Least resistance was found to imipenem. When compared to the sputum isolates, wound swab isolates were more resistant to the antibiotics used. Surprisingly, we found number of environmental isolates that were multidrug resistant which were resistant to cefetoxime, gentamicin, azetronam, and amikacin.
Figure 1: Resistance of the isolates for the antibiotics tested

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Identification of biofilm-associated genes

All the 30 isolates of sputum were positive for the biofilm-forming genes tested in this study. In the wound swab isolates, there was 100% presence of algD, pelA, pelD, and pslB genes, and only in one isolate, while there was an absence of pslA gene. Of the 60 clinical isolates, around 99% were positive for the genes tested. There was much variation in the presence of the biofilm genes in the environmental isolates. Prevalence of algD, pslA, pslB, pelA, and pelD was 47%, 74%, 80%, 50%, and 57%, respectively. In a greater number of environmental isolates, psl genes (pslA and pslB) were detected.

Quantification of biofilm

All the 90 isolates of P. aeruginosa were found to be biofilm formers. The average biofilm formed by P. aeruginosa isolates from sputum was more than the other isolates but less than the reference strain [Table 2]. The biofilm formed by the environmental isolates was more than the wound swab isolates but less than the sputum isolates. There was variation in the quantity of biofilm formed by the isolates. In the sputum isolates, 13.33%, 63.33%, and 23.33% were weak, moderate, and high biofilm formers, respectively. In the wound swab isolates, 10% were weak biofilm formers, 90% were moderate biofilm formers, and none of the isolates were high biofilm formers. In the environmental isolates, 30%, 56.66%, and 13.33% were weak, moderate, and high biofilm formers, respectively. There was presence of high biofilm formers in isolates from sputum and environment. Biofilm formation in clinical isolates was significantly higher than the environmental isolates (t = 0.386, P < 0.05).
Table 2: Biofilm formation in Pseudomonas aeruginosa isolates

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Three representative high biofilm formers from sputum (HS26), wound swab (HW20), and environment (EN4) were selected to check for their ability to form biofilm at different time points (18, 25, 48, and 72 h). There was an increase in the biofilm formed by these isolates at the end of 72 h [Table 3]. The highest biofilm formation was observed in the reference strain. The biofilm formed by the environmental isolate at the end of each time point was greater than the clinical isolates but less than the reference strain. There was a sudden increase in the biofilm formation in the wound swab isolate at 72 h, which was also encountered in the reference strain. The quantity of biofilm formed by the sputum and wound swab isolates at 72 h was nearly equal.
Table 3: Biofilm formation at different time points

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Quantification of biofilm genes in Pseudomonas aeruginosa isolates

Expression of pslA, pslB, pelD, and pelA was relatively lower than the reference strain at 18 h. PAO1 showed a nearly constant expression of pslA, pslB, pelD, pelA, and algD genes at different time points. At 25 h, there was a 2-fold increase in pslA, pelD, and pelA gene expression in the isolates of P. aeruginosa. In the environmental isolate, the relative expression of pslA, pslB, pelD, and pelA gene was increased by 11, 10, 10, and 9 folds, respectively, at 48 h. The expression of algD gene was significantly increased by 40 folds at 25 h and decreased drastically thereafter in the wound swab isolate. A 12-fold increase in the algD gene expression was observed in the environmental isolate at 48 h [Figure 2].
Figure 2: Relative expression of biofilm-forming genes in Pseudomonas aeruginosa isolates at different time points. (a) pslA; (b) pslB; (c) pelD; (d) pelA; (e) algD

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  Discussion Top


Infections with P. aeruginosa are difficult to eradicate because of their ability to form biofilms. These biofilms are not only less susceptible to host cell immune responses but also have a high tolerance to antibiotics than the planktonic cells.[4] Both clinical and environmental isolates of P. aeruginosa are known to possess biofilm-forming genes.[11] Although the genes involved in biofilm formation have been observed both in clinical and nonclinical P. aeruginosa isolates, there are limited studies on the expression of these virulence genes. Environmental and clinical isolates of P. aeruginosa are often differentiated from one another. Environmental isolates are generally considered to be susceptible to the antibiotics and due to their metabolic versatility adapt to degrade various carbon sources.[12] The clinical isolates evolve to cause infections and develop antibiotic resistance and lose their ability to utilize various carbon sources.[13] However, increase in the concentration of antibiotics in the environment has contributed to the appearance of multidrug-resistant strains in the nonclinical environment as well.[14] The present investigation explored the antibiotic susceptibility profile and the presence and expression of some of the virulence genes involved in biofilm formation in the isolates of P. aeruginosa recovered from different sites.

Antibiotic susceptibility profile of the clinical isolates revealed that none of the isolates showed complete susceptibility to the antibiotics used. The findings corroborate with a study which has reported high antibiotic resistance in clinical isolates.[15] As previously described, resistance or intermediate resistance was found in the environmental isolates against cephalosporins, carbapenems, penicillins, and fluoroquinolones.[12] In the current investigation, the environmental isolates were resistant to drugs other than the previously described antibiotics, indicating an increase in the range of resistance. The increase in drug resistance in environmental P. aeruginosa isolates is possibly due to a rise in the concentration of antibiotics in natural samples and also horizontal gene transfer in bacteria found within biofilms.[16]

Cell–cell and cell–surface interactions during biofilm formation in P. aeruginosa are due to the presence of pel, psl, and alg operons, which are responsible for the synthesis of extracellular polysaccharides.[17] Alginate, psl, and pelexo polysaccharides produced by P. aeruginosa have been proved to be involved in biofilm formation giving its characteristic architecture. Pel polysaccharide is important in the formation of static biofilms and is involved in later stages of biofilm maturity.[18] Psl role in the initial attachment of cells has been reported.[19] Alginate overproduction in P. aeruginosa results in an increase in the biofilm biomass, which facilitates competitive fitness during mixed species biofilm formation during chronic lung infection.[20]

In the current study, the biofilm-associated genes were found to be present in all the sputum isolates, indicating their possible role in biofilm formation in immunocompromised lungs, resulting in clinical treatment failure. Similarly, wound swab isolates also confirmed the presence of biofilm-associated genes, making these isolates more virulent during wound infections, leading to debilitating conditions. There was much variation in the presence of biofilm genes tested in the environmental isolates. In line with previous related studies, our findings demonstrated a greater prevalence of psl genes as compared to pel and algD indicating their vital role in attachment, leading to a mature biofilm.[21] All the isolates used in the present investigation were biofilm formers. The results are consistent with the data published in studies on P. aeruginosa that have proved biofilm formation in a majority of the clinical and environmental isolates.[22],[23] Quantity of biofilm formed in the environmental isolates (E1, E16, and E23) was very less when compared to other environmental strains. This might be due to the absence of pslA gene. The result supports a study that has shown the impairment in the Psl production by P. aeruginosa with the deletion of pslA, which ultimately impairs the surface attachment.[24] In the isolates that were considered as strong biofilm formers, the quantity of biofilm formed was greater, presumably due to the presence of pel genes. This finding is in agreement with a study that has shown increased Pel production which in turn increases cell–cell interactions and biomass of biofilms.[25] Increased antibiotic resistance in the clinical and environmental biofilm formers has been documented.[26],[27] Our findings also revealed the association between biofilm production and increased antibiotic resistance in the isolates used. PAO1, the laboratory strain, although originally isolated from clinical specimen, has been shown to form reduced biofilm as compared to fresh clinical and environmental isolates.[11] By contrast, the findings from the present study indicated more biofilm formation in PAO1 as compared to other clinical isolates at different time intervals. This suggests that PAO1 may be equipped with endogenous biofilm activation mechanisms, or conversely, other isolates used in the study possess mechanisms to suppress biofilm formation.

The relative expression of biofilm-associated genes in the clinical isolates tested was weak when compared to the environmental isolate. This kind of expression may be due to the environmental conditions in which bacteria are growing. It has been reported that the local environment dictates the bacterial responses which in turn regulate gene expression in biofilms.[28] The requirement of each of the three polysaccharides during biofilm formation varies in clinical and environmental isolates. As reported by Colvin et al.,[25] strains require at least one of the three polysaccharides for their biofilm formation, and in many strains of P. aeruginosa, psl and pel are functionally redundant in the biofilm matrix. A study by Colvin et al.[21] has classified the strains of P. aeruginosa based on their dependencies of biofilm formation. Accordingly, the environmental strain used for the expression studies is a class IV matrix producer as the expression level of psl and pel genes was very high. PAO1, sputum, and wound swab isolates are class III matrix producers as they produce relatively less amount of psl and pel. Although PAO1 has been reported to primarily rely on psl for biofilm formation, our expression data confirm its ability to transcribe and express some of the pel genes. The presence and expression of both psl and pel in all the four isolates suggest an advantage of having them in reducing the impact of mutations on one of the important functions such as biofilm formation.[21]

The expression of psl gene in the sputum isolate was less than the wound swab and environmental isolates. Previous reports have given evidence for the decreased production of Psl polysaccharide in the mucoid isolates isolated from the lungs and increased production of it in the nonmucoid strains.[29],[30] Psl has been identified as an adhesin in nonmucoid strains. The enhanced expression of pslA and pslB genes and the increased biomass in the nonmucoid environmental isolate were expected considering the over expression of Psl in nonmucoid isolates.[31] Production of alginate is thought to be a key polysaccharide in both mucoid and nonmucoid isolates of P. aeruginosa. It is the hallmark of a mature biofilm with increased resistance to antibiotics and antimicrobial stress.[32] In contrast to this study, a study by Stapper et al.[33] has shown low levels of alginate in nonmucoid isolates. The current investigation reveals the enhanced expression of alginate gene in nonmucoid isolates which also have shown increased resistance to the antibiotics used. Surprisingly, the alginate gene expression was very weak in the mucoid isolate from the lungs.


  Conclusion Top


The study infers the presence and expression of the biofilm-forming genes in the environmental isolate. It also suggests the possibility of these nonclinical ones becoming a clinical pathogenic one causing biofilm-associated infection. Knowledge gained from the present study sheds light on the virulence abilities of environmental P. aeruginosa isolates and also improvement in the existing approaches that target the prevention of transmission from the environmental sources. The study provides a better understanding of the phenotypic diversity found in clinical and environmental P. aeruginosa isolates in relation to biofilm formation.

Acknowledgment

We thank Nitte (Deemed to be University) for all the support provided.

Financial support and sponsorship

The work was supported by the Nitte (Deemed to be University) Grant number (NUFR2/2016/23-04).

Conflicts of interest

There are no conflicts of interest.



 
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