Assessment of pelA -carried Pseudomonas aeruginosa isolates in respect to biofilm formation

Owing to high antibacterial resistance of Pseudomonas aeruginosa , it could be considered as the main reason behind the nosocomial infections. P. aeruginosa has a well-known biofilm forming ability. The expression of polysaccharide encoding locus ( pelA gene) by P. aeruginosa is essential for this ability. The purpose of the current research was to determine the biofilm formation in P. aeruginosa isolated from clinical samples and to evaluate the role of the selected PelA gene in biofilm formation using PCR method in Iraqi patients. Results revealed that 24 (96%) isolates were found to have the ability to form biofilm that was remarkably related to gentamicin resistance. Moreover, the pelA gene was found in all biofilm-producers. In conclusion, the results of the current study revealed that the P. aeruginosa biofilm-producer isolates were resistant to the antibiotics in question. Likewise, because of wide spreading, it appears that the pelA gene is related to biofilm formation.


Introduction
removed by the ethanol in each well was directly quantified spectrophotometrically by measuring the OD 630 using microplate reader. The cut-off value (ODc) was calculated as three standard deviations plus the mean OD of the negative control. Given that, absorbance values represented the intensity of the biofilm formed by the studied isolates on the surface of the microtiter well. The obtained results were categorized into four groups (viz., non-biofilm producer, weak, moderate, and strong). Molecular study DNA of P. aeruginosa isolates was extracted and purified using Genomic DNA Extraction Kit (Promega, USA). The primers pairs used are designed to amplify a 118 bp fragment PelA gene by Colvin et al.2011 [18]. The primers pairs are pelA F: 5ˊ-CCTTCAGCCATCCGTTCTTCT-3ˊ and pelA R: 5ˊ-TCGCGTACGAAGTCGACCTT-3ˊ. PCR amplification was performed using Green master mix (Bioneer, Korea), 2 µl of 10 pmol/µl of each forward and reverse primers, 2 µl of template DNA (15-25 ng), volume was completed up to 20 µl with nuclease free water (Promega, USA). The PCR tubes containing the mixture were transferred to thermocycling (5 min at 94°C, 35 cycles of 30 sec at 94°C, 40 sec at 52°C, 50 sec at 72°C, and a final extension step 5 min at 72°C using Bio Rad, USA [19]. Thereafter, amplicons were resolved in 1.5% agarose gel.

Statistical Analysis
In order to determine the impact of parameters in this study using statistical package for social science (SPSS) 21.0 and Microsoft excel 2013. Categorical data formulated as count and percentage. Fisher exact test and chi square test was used to describe the association of these parameters. The lowest level of accepted statistical significant difference is bellow or equal to 0.01 [20].

Results Isolation and Identification
All the 60 isolates had a growth on nutrient, cetrimide agar and developed pale colonies on MacConkey agar and; nevertheless, they were oxidase and catalase positive. Hence, these isolates were primarily identified as P. aeruginosa (Table-1). Identification was confirmed using API 20E test.

Growth on Cetrimide agar
Growth with fluorescent green colour, elevated colonies, and grape-like odour

Growth at 42°C on Nutrient agar Positive
According to the specimen type, 60 isolates were categorized into seven groups, as shown in Table-2.

Antimicrobial susceptibility
The results showed highly significant (P≤0.01) variation in levels of resistance among the groups of antibiotics. The highest resistance results (68.3%) were recorded against each of Gentamicin and Ciprofloxacin. While the lowest resistance levels: 36.6% and 53.3% were against β-lactams represented by Ceftazidime and Imipenem, respectively (Figure-1).

Biofilm formation assay
This study showed a trend among the clinical isolates of P. aeruginosa to form a biofilm 24/25 (96%), except one isolate (4%) represented non-biofilm producer. Upon the criteria listed in Figure-2, the present study declared that three isolates (12%) formed weak biofilm, fourteen (56%) isolates formed moderate biofilm, whereas seven (28%) isolates formed strong biofilm.

Molecular study
PCR was carried out for the detection of PelA gene, which was involved in the formation of biofilm among the clinical isolates; which was expressed in all 25 tested isolates that developed a resistance to Gentamicin (Figure-3

Discussion
Pseudomonas aeruginosa is an opportunistic pathogen capable of causing a wide array of life threatening acute and chronic infections particularly in patients with compromised immune defense [21]. Earlier reports have shown that the antibiotic resistance of bacteria due to biofilm formation contributes to the persistence of bacterial cells and causes problems in the complete eradication of infection [22].
The variability in P. aeruginosa isolation percentage may be attributed to geographic, climatic, and hygienic factors among different areas. As well as, the high prevalence of P. aeruginosa in our community may be related to the rise of burn and wound patients than other samples in our population [23], which may be the result of different increased kitchen accidents, terrorist incidents, and electrical fires.
The differences in biofilm intensity among isolates of the present study might be due to several reasons; differences of isolates capacity to form biofilm or perhaps differences in primary number of cells that succeeded in adherence and differences of quality and quantity of quorum sensing signaling molecules (autoinducer) that produced from each isolate play important roles [24]. However, this high biofilm intensity may be related to susceptibility of microtiter plate assay in determining the few amounts formed, and is regarded as an essential method in studying the early stages of biofilm formation due to its use of constant conditions and it can be effective in studying different types of virulence factors to form biofilm such as pili, and flagella. In addition we can use this method with different types of bacteria [25].
On the other hand, Heydari and Eftekhar [26] indicated that the diversity of the ability of isolates to form biofilm is due to the association of production with its capability to produce different types of ß-Lactamase leading to forming a strong biofilm in comparison with the isolates that produced one type of enzyme. On the contrary, the isolates did not produce this enzyme were unable to form a biofilm. Besides, the variety in results might be due to the kind of media or the laboratory conditions that accompanied the detection of biofilm formation.
Numerous bacteria occur in turn between two forms of growth: planktonic cells and biofilms. These organized communities of bacteria are capable of growing on different surfaces and are the major factor of several diseases. Approximately, 80% of all microbial infections are related to biofilms [27]. Besides, several bacteria can form biofilms on abiotic surfaces. These microorganisms are usually considered as responsible for bio corrosion and biofouling in a variety of industry sectors. They may lead to the destruction of the equipment and accordingly to an increase in the maintenance costs [28].
The antibiotic susceptibility test was performed for aminoglycosides, fluoroquinolones, and beta lactam antibiotics with all the 60 isolates. Nearly 68.3% and 61.6% of isolates developed resistance toward gentamicin and amikacin, respectively. Starkey et al.2009 [29] reported that gentamicin in comparison to other aminoglycosides exhibited increased levels of resistance which may be due to the presence of PelA gene. Colvin et al. [18] assumed that pel is capable of providing preservation to planktonic cells when artificially overexpressed, thus suggesting that Pel plays an important protective role in biofilms of Pseudomonas strains. The aminoglycosides and fluoroquinolones antibiotics developed the highest resistance with respect to biofilm producing strains. Gentamicin and ciprofloxacin expressed 68.3% resistance. The carbapenems antibiotics revealed 53.3% resistant towards imipenem. The beta lactam antibiotics showed resistance lower level in biofilm producing strains; yet, ceftazidime expressed 36.6% resistance. Production of β-lactams enzymes in some isolates, which act to destroy β-lactams ring thereby leads to modification of antibiotics structure and spoilage their effects [30]. Moradali et al. [31] stated that P. aeruginosa is regarded as a main causative agent of nosocomial infections owing to its wide spectrum of antibiotic resistance. Saderi and Owlia [32] clarified that this ability either be normal or may be acquired through mutations in their genetic material or through the horizontal transference of genes.
In the present study, the isolates produced strong, intermediate and weak biofilms were screened for PelA gene, which is responsible for production of biofilm. Pel is essential for conserving cell-tocell interactions in P. aeruginosa biofilm, service as a primary structural frame for the community. It also plays a secondary role by increasing resistance to aminoglycoside antibiotics and this defense happens only in biofilm populations [33].
The current study observed that pelA gene was expressed heavily 25/25(100%) amongst the biofilm producing isolates. The phenotypically positive isolates for production of biofilms in vitro were genotypically positive for expression of PelA gene, while those isolates phenotypically non-producing biofilm and harboring PelA gene were also found to be resistant towards aminoglycosides [19]. This gene was found in almost all clinical strains of P. aeruginosa. The presence of this gene could not predict which strains will produce a biofilm because the biofilm formation is affected by many factors. On the other hand, there was no gene expression appear in some isolates, despite the formation of the biofilm in these isolates, this means a contribution of other genes that are not studied in current research has a role in the formation of the biofilm.

Conclusion
The current study showed that most of the biofilm formed isolates were highly resistant to even higher generations of aminoglycosides, fluoroquinolones and beta lactam group of antibiotics. It was also observed that most of the isolates showing resistance to aminoglycosides antibiotic carried PelA gene. Henceforth, more work is needed to fully elucidate the antibiotic resistance mechanisms in biofilms and develop new therapeutic strategies.