127J Contemp Med Sci | Vol. 9, No. 2, March-April 2023: 127–133 Original A Potential Role of Extracellular DNA in Biofilm and Ciprofloxacin Resistance Hind Tahseen Ibrahim1, Ali A. Mussa2, Harith Jabbar Fahad Al-Mathkhury2* 1Department of Medical Laboratory Techniques, College of Medical (Technology), Al-Farahidi University, Baghdad, Iraq. 2Department of Biology, College of Science, University of Baghdad, Baghdad, Iraq. *Correspondence to: Harith Jabbar Fahad Al-Mathkhury (E-mail: harith.fahad@sc.uobaghdad.edu.iq) (Submitted: 26 January 2023 – Revised version received: 19 February 2023 – Accepted: 03 March 2023 – Published online: 26 April 2023) Abstract Objectives: This study aims to broaden our knowledge of the role of eDNA in bacterial biofilms and antibiotic-resistance gene transfer among isolates. Methods: Staphylococcus aureus, E. coli, and Pseudomonas aeruginosa were isolated from different non-repeated 170 specimens. The bacterial isolates were identified using morphological and molecular methods. Different concentrations of genomic DNA were tested for their potential role in biofilms formed by study isolates employing microtiter plate assay. Ciprofloxacin resistance was identified by detecting a mutation in gyrA and parC. Results: The biofilm intensity significantly decreased (P < 0.05) concerning S. aureus isolates and insignificantly (P > 0.05) concerning E. coli isolates. Yet, one E. coli isolate’s biofilm was significantly decreased (P < 0.05) linearly with increasing eDNA. Of considerable interest, the addition of eDNA led to a significant increase (P < 0.05) in the biofilm of the two-tested P. aeruginosa isolates. Moreover, eDNA participated in transferring Ciprofloxacin resistance to the sensitive isolate when it presents in its biofilm. Conclusion: eDNA has a dual effect on bacterial biofilms either supportive or suppressive following bacterial species per se. Also, it seems to play an important role in antibiotic resistance within the biofilm. Keywords: eDNA, Biofilm, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa ISSN 2413-0516 Introduction Staphylococcus aureus inhabited approximately 30% of healthy people, mostly in the anterior nares. Nevertheless, it is also a leading cause of hospital-associated and community-associated bacterial infections in humans, associated with numerous mild skin and soft tissue infections and life-threatening pneumonia, aimeretcab, osteomyelitis, endocarditis, sepsis, and toxic shock syndrome. The increasing prevalence of methicillin-resistant S. aureus (MRSA) and its ability to resist multiple drugs has posed a serious challenge to infection control.1,2 Escherichia coli is one of the earliest colonizers of the gas- trointestinal tract; although eventually, it is a minor compo- nent of the colonic gut microbiome in humans, where it represents less than 0.1% of the total bacterial cells. Neverthe- less, due to the overall high cell density in the colon, this small percentage translates into around 108 cells/ml.3 Indeed, E. coli is the causative agent of various intestinal and extra-intestinal diseases, including being suspected to be the cause of sudden infant death syndrome.4,5 Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen and the leading cause of diverse nosocomial infections and it is commonly difficult to eradicate with conventional anti- biotic therapy, particularly when established as biofilms.6 Although P. aeruginosa rarely infects healthy people, those indi- viduals whose skin, mucous membranes, or immune system are affected, are more susceptible to becoming infected by this organism; for example, burn victims, patients with cystic fibrosis, or cancer patients treated with chemotherapy.7 Biofilms are surface-associated bacterial communities embedded in an extracellular matrix that is considered to be a major problem in the context of chronic infections because biofilm-dwelling cells have increased antibiotic resistance compared to their planktonic counterparts.8 The critical roles of the matrix for microbial interactions and virulence, as well as for antimicrobial tolerance, are being increasingly recognized. The matrix production enhances bacterial cell adhesion and cohesion (resulting in densely packed cell aggre- gates), providing mechanical stability.9 Extracellular deoxyribonucleic acid (eDNA) is widely rec- ognized as an integral component of biofilms’ extracellular polymeric matrix (ECM). Many studies mentioned that eDNA plays a pivotal role in bacterial biofilm formation. The involve- ment of eDNA in biofilms includes providing nutrition and energy for sessile cells promoting horizontal gene transfer (HGT) in naturally competent cells or maintaining the biofilm integrity.10 While others have proved that eDNA could desta- bilize the biofilm formation process and that effect would depend on the bacterial species or its serotypes.11 Upon the aforementioned facts, this study aimed at 1) investigating the effect of increasing concentration of eDNA on biofilm formation and 2) inspecting the transferring possi- bility of the antibiotic-resistant gene from eDNA to bacterial cell within the biofilm. Materials and Methods Ethical Statement This work is approved by the College of Science Research Ethics Committee (ref. CSEC/1220/0081). All participants agreed to provide the investigator with the specimens. Informed consent according to the Declaration of Helsinki was obtained from all participants. Specimen Collection A total of 170 different non-repeated specimens were collected from patients referring to hospitals in Baghdad, Iraq. These specimens comprised anterior nares swabs (n = 20) were taken from healthcare workers as well as the patients, sputum (n = 30), mid-stream urine (n = 95), burn swabs (n = 13), and mailto:harith.fahad@sc.uobaghdad.edu.iq 128 J Contemp Med Sci | Vol. 9, No. 2, March-April 2023: 127–133 A Potential Role of Extracellular DNA in Biofilm and Ciprofloxacin Resistance Original H.T. Ibrahim et al. blood (n = 12). The specimens were cultured on different selective culture media; Mannitol salt agar, MacConkey agar, Eosin Methylene Blue (EMB) Agar, and Cetrimide agar and subsequently subjected to conventional biochemical tests including Catalase, Oxidase, Coagulase, Acetoin production, IMViC, Motility, and Haemolysin Production Test) to identify Staphylococcus aureus, E. coli, and Pseudomonas aeruginosa, Salmonella enterica serovar Typhi and Klebsiella pneumoniae isolates.12 All the bacterial isolates were then tested for Cipro- floxacin resistance by measuring the minimal inhibitory con- centration for Ciprofloxacin using the Agar diffusion method following the method described by Jennifer.13 Polymerase Chain Reaction Bacterial genomic DNA was extracted using Presto™ Mini gDNA Bacteria Kit (Geneaid, Taiwan) and all amplifications were carried out using AccuPower® PCR PreMix, and Gradient master cycler (Eppendorf, Germany). All S. aureus-suspected isolates were screened for the presence of the S. aureus species-specific 16S rDNA gene using specific primers, SA1: (AATCTTTGTCGGTACACGATAT- TCTTCACG) and SA2: (CGTAATGAGATTTCAGTAGA- TAATACAACA) were used to amplify 108 bp segment of S. aureus species-specific 16S rDNA gene. The reaction protocol was as followed: Initial denaturation at 92°C for 3 min fol- lowed by 30 cycles of 92°C 1 min, 56°C 1 min, and 72°C 1 min; following that 3 min at 72°C for final extension.14 S. aureus iso- lates were also screened for methicillin resistance by detecting mecA gene using specific primers MecA1: (GTAGAAAT- GACTGAACGTCCGATAA) and MecA2: (CCAATTCCA- CATTGTTTCGGT); The reaction condition included initial denaturation at 94°C for 10 min followed by 10 cycles of 94°C 45 sec, 55°C 45 sec and 72°C 75 sec; followed by 25 cycles of 94°C 45 sec, 50°C 45 sec and 72°C 75 sec.15 Escherichia coli-suspected isolates were also screened for the presence of the uspA gene by the same technique employing specific primers uspA-F (CCGATACGCTGCCAATCAGT) and uspA-R (ACGCAGACCGTAGGCCAGAT), the condi- tions were: Initial denaturation at 95°C for 5 min followed by 30 cycles of 94°C 30 sec, 56°C 30 sec and 72°C 30 sec; fol- lowing that 5 min at 72°C for final extension.16 Two Ciprofloxacin-resistant S. aureus isolates were selected to detect any possible mutation in gyrA and parC coding for DNA gyrase subunit A and DNA topoisomerase IV, respectively using the specific primers. GyrA-F: AAATCTGCCCGTGTCGTTGGT and GyrA-R GCCAT- ACCTACGGCGATACC for gyrA; ParC-F: GTATGCGAT- GTCTGAACT and ParC-R TTCGGTGTAACGCATTGC for parC. The amplification program involved initial dena- turation at 95°C for 2 min followed by 35 cycles of 95°C 30 sec, 55.4°C 60 sec, and 72°C 60 sec.17 The sequences of the PCR products were obtained using the Sanger method and then were aligned with gene sequences from National Center for Biotechnological Information (NCBI) (https://www.ncbi.nlm.nih.gov/) to investigate for mutations. All Ciprofloxacin resistant isolates were screened for the presence of acrA gene coding for acrAB efflux pump. The primers that were used are AcrA-F: (ATGAACAAAAACA- GAGG) and AcrA-R: (TTTCAACGGCAGTTTTCG) in a PCR reaction program of initial denaturation at 94°C for 5 min followed by 30 cycles of 94°C 1 min, 52°C 1 min and 72°C 1 min followed by 5 min at 72°C for final extension.18 Biofilm Formation Assay Quantification of biofilm formation by E. coli, S. aureus, and P. aeruginosa on abiotic surfaces was assessed as previously described.19 In brief; wells of sterile 96-well U-shaped- bottomed polystyrene microplates were filled with 200 μl of an overnight TSB (bacteria concentration was adjusted to in equivalence to McFarland standard no. 0.5) before the plates were covered and incubated aerobically at 37°C for 24 h. Each bacterium was tested in triplicate. Control wells were per- formed by adding bacteria-free TSB. The wells were aspirated and washed three times with 200 μl sterile phosphate-buffered saline (PBS); the remaining attached bacteria were fixed with 200 μl methanol for 15 min. After drying in air, the wells were stained with 200 μl 0.1% crystal violet solution for 15 min at room temperature. The excess stain was rinsed off by placing the plate under running tap water. Thereafter, the plates were dried. Subsequently, the adherent cells were resolubilized with 200 μl of 33% glacial acetic acid for 15 minutes. Finally, the optical density (OD) of each well was obtained at 600 nm using a microplate reader (Biotek, UK). Cut off value (ODc) was calculated as the mean of OD of control wells plus 3 standard deviations. The isolates were then interpreted as Non–producer (OD ≤ ODc), weak producer (ODc < OD ≤ 2*ODc), moderate producer (2*ODc < OD ≤ 4*ODc), or strong producer (4*ODc < OD). To investigate the impact of eDNA concentration on bio- films of E. coli, S. aureus, and P. aeruginosa, the same protocol described previously was followed; nonetheless, different concentrations (400 ng/µl, 200 ng/µl, 100 ng/µl, and 50 ng/µl as a final concentration) of purified eDNA were added to each well separately. Moreover, 100 μl of TE buffer was added to the control wells instead of purified eDNA. Thereafter, plates were incubated, stained, and quantified as it is mentioned earlier. Determining the Role of eDNA in Gene Transfer An aliquot of 100 µl of the bacterial growth (compatible with McFarland standard no. 0.5) of Ciprofloxacin sensitive isolates of E. coli (E4), S. aureus (S4) and P. aeruginosa (P1) was added to wells of sterile 6-well U shaped-bottomed polystyrene microplates; thereafter, three ml of sterile tryptic soy broth were added to each well. A volume of one ml of eDNA (400 ng/µl) extracted from Ciprofloxacin-resistant isolate (S. aureus isolate S17) was added to each well. All plates were covered and incubated at 37°C for 24 h. then washed thrice with sterile PBS. Biofilms were removed from each well by scraping, sus- pended in a sterile broth medium, and incubated at 37°C for 18 h. The minimal inhibitory concentration to Ciprofloxacin was determined and further investigation was carried out using PCR technique for gyrA, parC, and acrA genes as it is mentioned previously, followed by sequencing of amplified products. Statistical Analysis Biofilm data were analyzed using two-way ANOVA followed by LSD0.05. The differences were considered significant when P < 0.05. https://www.ncbi.nlm.nih.gov/ 129J Contemp Med Sci | Vol. 9, No. 2, March-April 2023: 127–133 H.T. Ibrahim et al. Original A Potential Role of Extracellular DNA in Biofilm and Ciprofloxacin Resistance Results and Discussion identification results revealed that 25, 24, and 2 isolates were identified as S. aureus, E. coli, and P. aeruginosa, respectively. Furthermore, all S. aureus isolates were found to be methicillin- resistant due to harboring the mecA gene. The polymerase chain reaction was also employed to detect the presence of acrAB efflux pump using primers that are specific for acrA gene encoding for this pump in all Cipro- floxacin-resistant isolates (two S. aureus & 13 E. coli isolates). The result revealed the presence of a single gene with 495 bp in all of these isolates. The present results are in line with those obtained by Pakzad et al.18 in that all the resistant isolates har- bored the acrA gene. On the other hand, these results differ considerably from those reported by the same authors as they reported that not all Ciprofloxacin-sensitive isolates contained this gene. Detection of gyrA and parC Mutations Ciprofloxacin-resistant S. aureus isolates (S17 and S18) were carefully chosen to be investigated for mutations in gyrA and parC genes. Two specific sets of primers were used to amplify gyrA and parC genes in separate PCR reaction tubes; after electrophoresis of the products and illumination under UV light, specific bands were obtained at 344 and 230 bp for gyrA and parC, respectively. Such results were expected as these genes are considered to be part of the structural genes of the bacterial cell. The sequences of the PCR product of the isolate S17 were obtained and compared to sequences of gyrA and parC genes from NCBI; as illustrated in Table 1 and Table 2, about 40 and Table 1. List of mutations in gyrA forward strand No. Mutation Type No. Mutation Type 1 G®C Transversion 21 A®C Transversion 2 T®C Transition 22 C®T Transition 3 T®G Transversion 23 A®C Transversion 4 T®A Transversion 24 C®T Transition 5 G®- Deletion 25 T®A Transversion 6 A®C Transversion 26 C®T Transition 7 G®A Transition 27 G®A Transition 8 A®T Transversion 28 T®C Transition 9 A®C Transversion 29 A®G Transition 10 T®A Transversion 30 A®T Transversion 11 A®C Transversion 31 A®C Transversion 12 G®A Transition 32 C®A Transversion 13 C®T Transition 33 A®C Transversion 14 A®C Transversion 34 T®G Transversion 15 A®T Transversion 35 C®A Transversion 16 A®C Transversion 36 G®T Transversion 17 G®T Transversion 37 A®T Transversion 18 C®A Transversion 38 C®T Transition 19 G®A Transition 39 C®T Transition 20 A®T Transversion 40 G®C Transversion Table 2. List of mutations in gyrA reverse strand No Mutation Type No Mutation Type 1 A®– Deletion 27 T®G Transversion 2 T®– Deletion 28 A®G Transition 3 C®A Transversion 29 G®A Transition 4 G®T Transversion 30 A®G Transition 5 A®C Transversion 31 C®T Transition 6 G®T Transversion 32 C®T Transition 7 T®G Transversion 33 G®A Transition 8 T®A Transversion 34 A®T Transversion 9 A®G Transition 35 T®G Transversion 10 T®C Transition 36 C®T Transition 11 A®G Transition 37 A®T Transversion 12 C®T Transition 38 A®T Transversion 13 G®A Transition 39 G®T Transversion 14 A®T Transversion 40 T®G Transversion 15 G®A Transition 41 T®G Transversion 16 T®C Transition 42 T®A Transversion 17 G®A Transition 43 A®T Transversion 18 T®C Transition 44 G®A Transition 19 A®C Transversion 45 A®T Transversion 20 C®T Transition 46 A®G Transition 21 G®T Transversion 47 G®A Transition 22 T®G Transversion 48 T®C Transition 23 T®G Transversion 49 T®G Transversion 24 T®A Transversion 50 T®A Transversion 25 T®G Transversion 51 G®A Transition 26 G®A Transition 51 mutations in the forward and reverse strands, respectively, were detected in gyrA of the tested isolate; since gyrA encodes for DNA gyrase, these mutations while leading to amino acid substitutions, alter the target protein for fluoroquinolone structure and subsequently the fluoroquinolone binding affinity of the enzyme, leading to drug resistance.20 On the other hand; after comparing the obtained sequence of parC from the tested isolate with sequences from NCBI, the result revealed complete similarity, and no mutations were recorded. In plain words, resistance to Ciprofloxacin in the tested isolates is due to a mutation in gyrase rather than topoi- somerase IV. Biofilm Formation Assay The Microtiter plate assay is the most widely used and was considered a standard test for the detection of biofilm forma- tion. This method has been reported to be the most sensitive, accurate, and reproducible screening method for the determi- nation of biofilm production by clinical isolates of S. aureus, E. coli, and P. aeruginosa and has the advantage of being a quan- titative tool for comparing the adherence of different strains.21 130 J Contemp Med Sci | Vol. 9, No. 2, March-April 2023: 127–133 A Potential Role of Extracellular DNA in Biofilm and Ciprofloxacin Resistance Original H.T. Ibrahim et al. The result revealed that only 8% of S. aureus isolates were strong biofilm producers; while 60% and 32% of the isolates were moderate and weak producers, respectively. On the other hand, none of the tested E. coli isolates were strong biofilm producers; whereas 68% and 32% of the isolates were mod- erate and weak producers, respectively. Similar trends have been reported by Mohammed et al.22 in that 14% of their local S. aureus isolates were strong bio- film-producers, 43% were low biofilm intensity and 43% were biofilm-negative. Mathur et al.21 similarly conclude from their data that about 14.47% and 39.4% of S. aureus isolates exhib- ited high and moderate biofilm formation, respectively; while 46% were weak isolates. These data are not consistent with those reported by Saeed et al.23 who stated that about 12.5% of isolated local strains of E. coli were strong biofilm producers while it agreed partially with their findings in that 87.5% of E. coli were moderate biofilm producers. It also disagrees greatly with Fattahi et al.24 who found 38% of E. coli isolates were strong biofilm producers while 22%, 32%, and 8% of the iso- lates were moderate, weak, and non-biofilm producers respec- tively. The results are generally consistent with the findings of Ghafil25 in that the ability of S. aureus to form biofilm was higher than that of E. coli. S. aureus biofilms, once established, are recalcitrant to antimicrobial treatment and the host response, and therefore are the etiological agent of many recurrent infections that have a demonstrated biofilm component.26 Chronic infections are associated with the biofilm mode of growth where S. aureus can attach and persist on host tissues, such as bone and heart valves, to cause osteomyelitis and endocarditis respectively, or on implanted materials, such as prosthetic joints,27 catheters,28 Table 3. Impact of eDNA on biofilm Isolate code OD 600 P-value LSD 0.05Control (No eDNA) 50 ng/µl of eDNA 100 ng/µl of eDNA 200 ng/µl of eDNA S1 0.151 0.114 0.108 0.112 0.004000 0.018 S2 0.181 0.152 0.149 0.113 0.000024 0.012 S3 0.193 0.182 0.154 0.167 0.016000 0.020 S4 0.178 0.133 0.143 0.134 0.035000 0.027 S5 0.151 0.150 0.133 0.127 0.160000 – E1 0.135 0.124 0.122 0.112 0.246066 – E2 0.131 0.133 0.118 0.109 0.204402 – E3 0.166 0.178 0.204 0.159 0.221004 – E4 0.142 0.173 0.164 0.151 0.501460 – E5 0.148 0.131 0.119 0.099 0.000206 0.011 P1 0.143 0.156 0.220 1.036 0.000001 0.012 P2 0.112 0.111 0.124 0.138 0.000010 0.004 K1 0.097 0.130 0.235 0.751 0.000004 – K2 0.113 0.171 0.185 0.217 0.000024 – Se1 0.112 0.134 0.115 0.109 0.006000 0.019 Se2 0.154 0.162 0.142 0.126 0.033009 – Se3 0.164 0.162 0.159 0.148 0.449729 – Se4 0.150 0.142 0.127 0.116 0.084083 – Se5 0.301 0.291 0.289 0.266 0.399151 – and pacemakers.29 Chronic S. aureus infections that are associ- ated with biofilm frequently lead to significant increases in both morbidity and mortality, mainly when the infection is associated with indwelling medical devices.30 Implanted mate- rials become coated with host proteins upon insertion, and the matrix-binding proteins on the surface of S. aureus facilitate attachment to these proteins and the development of a biofilm. In cases of infected medical devices, removal of the device is often necessary to treat the infection.31 Complications in E. coli-related infection have been mainly attributed to biofilm formation. E. coli biofilm forma- tion is an intricate process that involves several steps such as initial adhesion, early development, maturation, and disper- sion. These steps are governed by many genes that serve spe- cific functions in the formation of the biofilm. E. coli biofilm has frequently been resistant to numerous antibiotics, mostly accredited to putative multidrug resistance pumps. The devel- opment of the extracellular matrix and the observed increased resistance to common antibiotics create a challenge to control the infections caused by E. coli biofilms.32 Impact of eDNA Concentration on Biofilm Intensity To investigate the impact of eDNA on biofilm, Different con- centrations of genomic DNA were added to the wells of a microtiter plate containing selected bacterial isolates of the species E. coli, S. aureus, P. aeruginosa, Salmonella enterica ser- ovar Typhi and Klebsiella pneumonia. The result presented in Table 3 revealed that the addition of increasing concentrations 131J Contemp Med Sci | Vol. 9, No. 2, March-April 2023: 127–133 H.T. Ibrahim et al. Original A Potential Role of Extracellular DNA in Biofilm and Ciprofloxacin Resistance From Figure 1, it can be noted that only 8% of S. aureus isolates were resistant to Ciprofloxacin; whereas 28% devel- oped intermediate resistance and 64% were sensitive to this antibiotic. On the other hand, about 44% of E. coli isolates were resistant to Ciprofloxacin while no intermediate resist- ance was observed among the tested isolates; nevertheless, 56% were sensitive. Regarding P. aeruginosa, the two tested isolates were Ciprofloxacin-sensitive. These findings confirm those of earlier studies, such as Mohamed et al.37 who found that the resistance of locally iso- lated E. coli strains from Iraqi patients to Ciprofloxacin was about 40.7%. Whereas they differ slightly from those reported by Al-Jebouri and Mdish38 who found that only 25% of E. coli and 40% of S. aureus isolates were resistant to Ciprofloxacin. Furthermore, our findings are in good agreement with Al-Marjani et al.39 who stated that about 16% of S. aureus iso- lates were resistant to Ciprofloxacin. The increasing resistance of bacteria to Ciprofloxacin could probably be augmented by using it to treat many infec- tions including prostatitis, UTI, endocarditis, gastroenteritis, infections of bones and joints, lower respiratory tract infec- tion, and enteric fever, among others, even though the risk of tendon rupture could increase upon using it. Notably, another factor contributing to the problem is the availability of Cipro- floxacin as an oral suspension that is currently flooding the market; even though, it is not licensed by the FDA to treat chil- dren with Ciprofloxacin due to the high risk of permanent injury to the musculoskeletal system except for inhalation anthrax and cystic fibrosis.40 Determining the Role of eDNA in Gene Transfer This experiment was designed to assess the possible role of eDNA in the transfer of antibiotic-resistance genes. Since the addition of eDNA has increased the biofilm intensity of P. aeruginosa isolate P1 only, our study was focused on that iso- late. The MIC of Ciprofloxacin was measured before and after the growth of the sensitive isolate in the presence of the DNA of Ciprofloxacin-resistant isolate (S. aureus isolate S17), the result revealed that the MIC value increased significantly (P < 0.05) from 1 to 4 µg/ml turning the bacterial isolate from sen- sitive to resistant to Ciprofloxacin. The acquired resistance was also tested after three successive generations and it was shown that the MIC value remained at 4 µg/ml. The same experiment was repeated using Ciprofloxacin-sensitive E. coli E4 and S. aureus S4 isolates as a recipient for gene transfer. Nonetheless, when measuring the MIC values before and after the gene transfer, it remained at 1 µg/ml; hence, the isolates of eDNA resulted in a significant decrease (P < 0.05) in biofilm intensity for the majority of the tested S. aureus isolates. S1-S5: S. aureus isolates 1-5; E1-E5: E. coli isolates 1-5; P1 and P2: P. aeruginosa isolate 1 and 2; K1 and K2: Klebsiella pneumoniae isolates; Se1-Se5: S. Typhi isolates Moreover, the biofilm intensity significantly decreased (P < 0.05) linearly with increasing concentrations of eDNA; on the other hand, although eDNA addition had led to thinner bio- film in the tested E. coli and S. Typhi isolates, the increasing concentration did not have a significant effect (P > 0.05) on the biofilm intensity; nevertheless, the biofilm of one strain of E. coli and two strains of S. Typhi was significantly decreased (P < 0.05) linearly with increasing eDNA. Surprisingly, the addition of eDNA led to a significant increase (P < 0.05) in the biofilm of the tested isolates of P. aeruginosa and K. pneumonia. The findings of this study agreed with the findings of Berne et al.33 who had informed that the biofilm formation of Caulobacter crescentus is significantly inhibited by the pres- ence of eDNA. Those results suggested that the bacteria would probably have a better chance for attachment to abiotic surfaces in the presence of DNase I, hence their ability to form more com- pact biofilm would increase; additionally, the biofilm forma- tion in Salmonella had significantly been inhibited upon the addition of exogenous eDNA. Another study conducted by Özdemir et al.34 revealed that eDNA could either enhance or decrease the biofilm formation by Salmonella and such effect of eDNA would be reliant on Salmonella serotype. Other studies that were conducted on the biofilm of Listeria monocytogenes and Neisseria meningitides come in contrast to our findings in which the biofilm formation had not been significantly affected by the addition of purified eDNA. However, crude extracts of eDNA in combination with probably some specific proteins or cell wall fragments promote the process of biofilm formation.35,36 Due to the interaction of eDNA with one or more of the biofilm components needs further investigation. inhibiting role of eDNA in the biofilm development of either S. aureus, E. coli or S. Typhi from our findings was Another study car- ried out by Wang et al.11 demonstrated the inhibitory effect of eDNA on Salmonella enterica biofilm who stated that Salmo- nella strains formed a thicker layer of biofilm in the presence of DNase I. of C. crescentus, which prevented the cells from settling into and encouraged the dispersal of cells. Determination of Minimal Inhibitory Concentration (MIC) Using Agar Diffusion Method The susceptibility of the bacterial isolates (S. aureus, E. coli, and P. aeruginosa) towards Ciprofloxacin was tested by deter- mining the MIC using the agar diffusion method. From the findings of the present study, various levels of susceptibilities to Ciprofloxacin among isolates were observed. The results are summarized in Figure 1. Fig. 1 Susceptibility of bacterial isolates to Ciprofloxacin. 132 J Contemp Med Sci | Vol. 9, No. 2, March-April 2023: 127–133 A Potential Role of Extracellular DNA in Biofilm and Ciprofloxacin Resistance Original H.T. Ibrahim et al. remained sensitive to Ciprofloxacin; consequently, no gene transfer occurred. The sequence analysis of the gyrA gene for the isolate before and after the addition of eDNA revealed slight variation, which furthermore confirms that the gene transfer process might have occurred and eDNA was respon- sible for that process. Correspondingly, no PCR product was obtained when trying to amplify the acrA gene after the gene transfer which implies that the acrA gene has not been trans- ferred during the process. The pool of eDNA found in bacterial biofilms provides a rich substrate for naturally occurring genetic transformation, which is the only alternative to mobile genetic elements and bacteriophage-induced gene transfer. This observation led to investigations into the role of DNA donor cells in biofilms and the conclusion that biofilm cells actively donate DNA to their prokaryotic neighbors.41 Extracellular DNA active in the nat- ural transformation was shown to be released by both Gram-positive and Gram-negative members of soil bacteria, thereby facilitating naturally occurring genetic transforma- tion. Natural habitats suitable for horizontal gene transfer are not limited to the soil. The majority of bacterial populations on earth are accompanied by eDNA, and it is known that such eDNA is suitable for horizontal gene transfer.42 Furthermore, it is well established that gene transfer occurs with enhanced efficiency in biofilms.43-46 Such horizontal gene transfer is facilitated by a biofilm lifestyle, which is characterized by cohabitation in close vicinity. This sharing of genetic material may function as the prokaryotic equivalent to sexual selec- tion,47 leading to beneficial adaptations such as antibiotic resistance48 or pathogenicity.49 Conclusion Extracellular DNA has a major role in the gene transfer pro- cess to biofilms. Given that, the addition of increasing con- centrations of eDNA resulted in a significant decrease (P < 0.05) in biofilm intensity for the majority of the tested S. aureus and E. coli isolates. Whereas, it has led to a significant increase (P < 0.05) in the biofilm of the two tested P. aerugi- nosa isolates. 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Mol Microbiol. 2001; 41: 263–277. doi:10.1046/j.1365-2958.2001.02520.x This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v9i2.1338