Study of Biolm Formation and Antibiotic Resistant Prole in Multidrug Resistant and Extensive Drug Resistance Pseudomonas Aeruginosa Isolated from Burn Wound Infections in Southwest Iran

Background: Pseudomonas aeruginosa is an opportunistic pathogen that has remained on the ‘top 10’ common hospital ‘superbugs’ worldwide for more than a decade. Study of biolm formation and antibiotic resistant prole in multidrug resistant and extensive drug resistance P. aeruginosa isolated from burn wound infections in southwest Iran Methods and Results: This study, which was performed in 110 P. aeruginosa isolates culture-positive reports. Assessment of biolm formation via microtiteplate and congo red agar. Overall, 110 clinical P. aeruginosa isolates were conrmed from wound burn infections. The maximum resistance rate among P. aeruginosa isolates to antibiotics tested was as follow Piperacillin, ceftazidime, and minimum resistance rate among P. aeruginosa isolates to antibiotics tested such as ticarcillin-clavulanic acid. The isolates were then evaluating the MICs by using the E-test. only 7 isolates were conrmed as colistin-resistant. Colistin reference MICs for the The prevalence of MDR P. aeruginosa was 38% and XDR- P. aeruginosa was 22% respectively. One of P. aeruginosa isolates were PDR. In microtiteplate assay,76% of the isolates have ability for biolm, formation, 40% were categorized as strong biolm-formers; 32% were moderate; 21% were weak biolm formers and 43% could not form any detectable biolm. Conclusion: in our study development of resistance by P. aeruginosa to many antimicrobial agents is a great challenge in controlling its infections. Therefore, the transmission of these isolates to patients leads to higher resistance. Therefore, the necessary hygiene measurements should be taken for the prevention of transferring the P. aeruginosa isolates to hospitalized patients. the majority of these deaths occur in low- or middle-income developing countries. (2) The mortality rate reported by different studies in Iran varies from 1.4/100,000 to 9.7/100,000 and regardless of study population types, case-fatalities have been variably reported in a range of 2–98%. (3) Damage to the skin barrier, larger burns, immunosuppression caused by burns, and prolonged hospital stay are important risk factors for infection. The type and size of the wound and the type and number of microorganisms. (4) Pseudomonas aeruginosa as one of the main causes of burn infections is responsible for an extensive range of serious infections (5). P. aeruginosa is a conditional pathogenic microorganism and has been among the top ten most common hospital "super bacteria" in the world for more than a decade. Therapeutic alternatives are nonetheless seriously constrained because of unfold of antimicrobial resistant lines; thus, P. aeruginosa contamination stays a life-threatening complication The up-law of innate resistance mechanisms along with overexpression of eux pumps and the purchase of overseas genetic determinants along with plasmids are crucial traits for the survival of P. aeruginosa at some stage in environmental pressures along with clinic environments. (7) The developing worldwide emergence of P. aeruginosa lines immune to all β-lactam sellers highlights the cap potential of this microorganism to evolve hastily to selective environmental stresses.(8) Multi-drug resistance of P. aeruginosa is diagnosed to be the various maximum hard antibiotic-resistant gram-poor bacilli to manipulate and treat.(9) Serious P. aeruginosa infections, each acute and chronic, are regularly nosocomial and related to compromised host defenses; Once an opportunistic pathogen like P. aeruginosa enters the its capacity to motive contamination has additionally been correlated with its tendency to shape biolms (10). Biolm formation is a procedure wherein microorganisms to and develop on a oor and bring extracellular polymers that facilitate attachment and matrix formation, ensuing in an the study is Study of biolm formation and antibiotic resistant prole in multidrug resistant and extensive drug resistance P.aeruginosa isolated from burn wound infections in southwest Iran. and 5(cid:0)-ACGGATCATCATCTTCTCC-3(cid:0).


Introduction
Burn patients are one of the most serious patients that doctors can see in their careers. These patients are in a dangerous period of various organ damage, in the acute phase; infection is the main cause of death. More than 75% of burn deaths in patients with severe burns are related to sepsis, infectious complications, and respiratory trauma. (1) Around 180,000 deaths occur annually due to burns, and the majority of these deaths occur in low-or middleincome developing countries. (2) The mortality rate reported by different studies in Iran varies from 1.4/100,000 to 9.7/100,000 and regardless of study population types, case-fatalities have been variably reported in a range of 2-98%. (3) Damage to the skin barrier, larger burns, immunosuppression caused by burns, and prolonged hospital stay are important risk factors for infection. The type and size of the wound and the type and number of microorganisms. (4) Pseudomonas aeruginosa as one of the main causes of burn infections is responsible for an extensive range of serious infections (5). P. aeruginosa is a conditional pathogenic microorganism and has been among the top ten most common hospital "super bacteria" in the world for more than a decade. Therapeutic alternatives are nonetheless seriously constrained because of unfold of antimicrobial resistant lines; thus, P. aeruginosa contamination stays a life-threatening complication (6). The up-law of innate resistance mechanisms along with overexpression of e ux pumps and the purchase of overseas genetic determinants along with plasmids are crucial traits for the survival of P. aeruginosa at some stage in environmental pressures along with clinic environments. (7) The developing worldwide emergence of P. aeruginosa lines immune to all βlactam sellers highlights the cap potential of this microorganism to evolve hastily to selective environmental stresses.(8) Multi-drug resistance of P. aeruginosa is diagnosed to be the various maximum hard antibiotic-resistant gram-poor bacilli to manipulate and treat.(9) Serious P. aeruginosa infections, each acute and chronic, are regularly nosocomial and related to compromised host defenses; Once an opportunistic pathogen like P. aeruginosa enters the host, its capacity to motive contamination has additionally been correlated with its tendency to shape bio lms (10). Bio lm formation is a procedure wherein microorganisms irreversibly connect to and develop on a oor and bring extracellular polymers that facilitate attachment and matrix formation, ensuing in an alteration with inside the phenotype of the organisms for increase price and gene transcription (11). The aim of this study is Study of bio lm formation and antibiotic resistant pro le in multidrug resistant and extensive drug resistance P.aeruginosa isolated from burn wound infections in southwest Iran.

Ethics declaration
The study was approved by the Research Ethics Committee (REC) of the Ahvaz Jundishapur University of Medical Sciences (No: IR. AJUMS.RE .1399.669) Iran. As the study was an observational one and did not involve any new intervention, it was conducted by the Declaration of Helsinki of 1975.

Specimens collection and Microbiological procedures
This study, which was performed in Taleghani Burn Center University Hospital, which is the major burn center in Ahvaz, Iran, from May 2019 to November 2020. One hundred and ten P. aeruginosa strains (replica isolates aren't included) culture-advantageous reviews consisting of wound 345 sufferers have been included. Patients who had an extended or admitted medical institution live because of burning infections with a hospitalization length of equal, extra than forty-eight hours have been studied. Patients have been excluded with any records of cancer, way of life-negative, immunocompromised diseases, and the usage of antibiotic tablets on the time of admission. Before present process wound excisions, the injuries of hospitalized sufferers have been controlled with topical 1% (Silver sulfadiazine) SSD cream with cumbersome dressings and modi ed each 24 hours. Samples have been accumulated from burn wounds via way of means of way of life swabs below aseptic techniques. According to the American Burn Association, wound colonization is described because the presence of a low awareness of microorganism at the oor without invasion or systemic manifestations. When there are greater than 10 ve bacterial isolates of tissue way of life with inside the wound, we name it a wound infection. When greater than 10 ve bacterial isolates of tissue way of life with inside the burn wound reasons the formation of pus and separation of the eschar, lack of graft with the involvement of tissue, or the presence of systemic sepsis, then it's far referred to as invasive infection (12). The samples were transferred to the laboratory immediately to the Department of Microbiology of Ahvaz Jundishapur University of Medical Sciences. P. aeruginosa isolates were con rmed by conventional and biochemical tests such as: gram stain, triple sugar iron (TSI), MRVP, oxidation fermentation (OF) test, citrate test, and pigment production in Mueller Hinton Agar (Biolife, Italia) and growth at 42°C. (13) The gram negative rod shape, MRVP negative, oxidative positive, cirate positive, triple sugar iron (TSI) agar reaction of alkaline over no change, growth at 42°C and production of bright-blue to blue green diffusible pigment on Mueller-Hinton agar.
Con rmation of P. aeruginosa -speci c PCR assay The boiling technique turned into used to extract genomic DNA from P.aeruginosa isolates. A few bacterial colonies of P.aeruginosa lines grown in a single day on nutrient agar (Merck, Germany) have been suspended in micro tubes containing 500 µl of Tris-EDTA buffer, then the micro tubes have been located in Incublock micro tube incubators (Denville Scienti c, USA) for ve min at 95°C, after which centrifuged at 14,000 rpm for 10 min at 4°C. The supernatant turned into used because the DNA template with inside the PCR assays. The DNA amount and ne have been assessed the usage of Nano Drop Spectrophotometer PROMO (Thermo Scienti c, USA) and electrophoresis on. ve% gel agarose, respectively. P.aeruginosa ATCC 27853 stress turned into used as a fantastic manage and distilled water as a terrible manage in all PCR reactions. Ampli cation turned into done the usage of an Applied Biosystems Veriti™ Thermal Cycler (Thermo Fischer Scienti c, Waltham, MA, USA) with the subsequent PCR conditions: preliminary denaturation at 95°C for three min, 25 cycles of 95°C for 20 s, 55°C for 30 s, and 65°C for two min, observed through a very last extension at 65°C for ve min. The following primers have been used: forward, lasI-

Minimum inhibitory concentration (MIC)
Colistin MIC have been measured through E-Test strips (Lio lchem, Italy) and interpreted primarily based totally on (CLSI 2020) guidelines. After measuring the MIC with the E-Test method, the P. aeruginosa isolates with a MIC Equal to and decrease than 2 µg/ml have been taken into consideration as "intermediate" and MIC Equal to and better than four µg/ml have been taken into consideration as "resistant". Escherichia coli ATCC 25,922 turned into used as great manipulate for antimicrobial susceptibility testing.

Assessment of bio lm formation via phenotype method
Bio lm formation in 96-well microliter plate(MTP) Bio lm production was determined by the MTp method as described previously (15). Brie y, overnight grown bacteria in trypticase soy broth (TSB) containing 0.25% glucose were diluted (1:100) and 200 µl portions were inoculated into 96-well at bottom polystyrene microtiter plates (Cellstar, greiner bio-one).
Incubation was carried out at 37 o C for 22-24 hr before removal of the cultures. The wells were washed 3 times with phosphate buffered saline (PBS, pH, 7.2), air dried and stained with 0.1% safranin. The optical density of the wells was measured at 490 nm using micro Elisa auto reader (Stat Fax 2100, Awareness Technol. Inc). An optical density of 0.12 was chosen to distinguish bio lm producers. Based on the calculation method described by Zhang et al., a cut-off optical density (ODc) was de ned as three standard deviations (SD) above the mean optical density (OD) of the negative control (inoculated medium): ODc = average OD of negative control+(3×SD of negative control). ODc value was calculated for each microtiter plate separately. For interpretation of the results, strains were divided into the following categories: OD ≤ ODc = non-bio lm producers; ODc (17) Congo red agar test Freeman et al. described a qualitative assay to detect the microorganisms which are able to produce bio lm. This method is based on color changing of colonies on the Congo red agar (CRA) medium. Colonies with black color represent a bio lm producer whereas red-pink colonies retain non-bio lm producers.

Data analysis
Chi-squared test was performed on the relationship between categorical variables, including bio lm characteristics and antimicrobial resistance using SPSS software, 22 (SPSS Inc., Chicago, IL, USA). A p-value of < 0.05 was considered signi cant.

Isolation of P. aeruginosa isolates and patient demographics
Overall, 110 clinical P. aeruginosa isolates were con rmed from wound burn infections. All P. aeruginosa isolates detected in biochemical tests were also con rmed by the molecular method. The mean age of the patients in the study was 35.32 years and (SD) was 11.74 years. (Table 1).
Antibiotic resistance in P. aeruginosa isolates The maximum resistance rate among P. aeruginosa isolates to antibiotics tested was as follow piperacillin 68% (n = 75), ceftazidime 59% (n = 65), meropenem 56% (n = 63), gentamycin 56% (n = 63), gati oacin 56% (n = 63) and minimum resistance rate among P. aeruginosa isolates to antibiotics tested such as ticarcillin-clavulanic acid 22% (n = 25), and ceftolozane-tazobactam 16% (n = 6) ( Table 2). These isolates were then evaluating the MIC by using the E-test. only 7 isolates were con rmed as colistin-resistant. Colistin reference MICs for the 110 P. aeruginosa bacteria were from 6 to 128 mg/L The incidence of MDR and XDR in P. aeruginosa isolates The incidence of MDR P. aeruginosa was 38% (n = 42), and XDR-P. aeruginosa was 22% (n = 25) respectively. One of P. aeruginosa isolates were PDR. The phenotypic antibiotic resistance pattern, prevalence, and diversity of the 110 P. aeruginosa isolates from the wound are shown in Table 1. The table reveals, that 74 different combination patterns were ranging from thirteen antibiotics in each combination. (Table 3) Out of the 110 P. aeruginosa isolates, one isolate was resistant to the 7 antibiotics representing thirteen classes.
The evolution of bio lm production in P. aeruginosa isolates The bio lm manufacturing evaluation through MTP and Congo pink agar methods. In the CRA method, 66(67%) isolates have been taken into consideration as generating bio lm and produced black colonies while 44 (50%) isolates produced pink colonies. In MTP differentiated isolates into sturdy, mild, susceptible, and no bio lm-forming in step with the OD values at 570 nm for (as effective control) and TSB (as poor control) have been 0.525 ± 0.062 and 0.055 ± 0.009, respectively. The OD570 values for the medical lines ranged from 0.125 ± 0.056 to 1.745 ± 0. 054.Again, 76% (n = 84) of the isolates have capacity for bio lm, formation, 40% (n = 45) have been classi ed as sturdy bio lm-formers; 32% (n = 19) have been mild; 21% (n = 20) have been susceptible bio lm formers and 43% (n = 26) couldn't shape any detectable bio lm. In the forty-ve sturdy bio lm-manufacturer, 76% (n = 9) isolates have been XDR and 76% (n = 18) isolates have been MDR and 0ne remoted turned into PDR. Among the 76% (n = 19) mild bio lm formers, 76% (n = 9) isolates have been XDR and 76% (n = 6) isolates have been MDR. Also, of the 20 susceptible bio lm-formers, 76% (n = 3) isolates have been XDR and 76% (n = 8) isolates have been MDR (Table 2). According to results, antibiotic resistance in P. aeruginosa non-bio lm manufacturer is better than P. aeruginosa isolates bio lm manufacturer ( Fig. 1 and table4). The statistical evaluation of the no courting among bio lm-forming capacity and antibiotic resistance (P = 0.781). However, the statistical evaluation of the connection among bio lm-manufacturer capacity and XDR (P = 0.042). Discussion P. aeruginosa is one of the main etiological agents of wound infections. In our study, the prevalence of P. aeruginosa in burn wound was 31%. In previous studies, in our region P. aeruginosa in burn wound were found to be 44%% to 69.9%. (19,20) The rate of P. aeruginosa vary between different in our region, which may be due to the performance of various infection control procedures. today, drug resistance is a massive developing trouble in treating infectious illnesses. The effects of us examine con rmed that 38% of P. aeruginosa isolates had been MDR. The excessive incidence of MDR P. aeruginosa isolates turned into additionally suggested from a few research in Iran and different countries. (21,22) The multidrug antibiotic resistance can lower the e cacy of the not unusual place antibiotics used with inside the scienti c putting specially with inside the infections. During the current decades, the carbapenems have encouraged because the rst-line antibiotics for the remedy of P. aeruginosa infections. However, unfortunately, growing resistance to carbapenems has been suggested global amongst P. aeruginosa isolates. (23) In us examine, the antibiotic susceptibility takes a look at effects con rmed that almost all of those isolates had been proof against meropenem (57%), imipanem (47%) and doripenem (67%). In settlement with ours, the excessive quotes of carbapenemresistant and doripenem. P. aeruginosa isolates had been suggested from different research. (24-26) Also, our effects con rmed that the antibiotic resistance quotes to gentamycin, amikacin, tobramycin, piperacillin, aztreonam, imipenem, meropenem, ceftazidime, gati oxacin, and nor oxacin amongst MDR P. aeruginosa isolates had been extra than 50%. In much like our work, Perez et al, Del Barrio et al additionally, suggested the excessive incidence of the resistance to those antibiotic dealers amongst P. aeruginosa isolates. (27,28) Colistin are the most effective remedy alternatives for infections as a result of appreciably drug resistant (XDR) or MDR A. P. aeruginosa. However, this antibiotic has a few unwanted facet effects, along with nephrotoxicity and neurotoxicity. (29) The antibiotic susceptibility takes a look at effects con rmed that almost all of P. aeruginosa isolates had been liable to colistin which can be in settlement with different reviews acquired from preceding research in Iran and different countries. (30)(31)(32) Hence, those effects endorse that colistin remains the only antibiotic dealers in opposition to MDR P. aeruginosa isolates. The bio lm matrix can extensively defend microorganism from each the immune device cells and antibiotic dealers. Bio lm formation of P. aeruginosa ends in lack of antibacterial susceptibility and the usage of extra concentrations of antibiotics with inside the remedy of infections as a result of such isolates. In us examine, maximum P. aeruginosa isolates had the cappotential of bio lm manufacturing however with unique capacities. As stated above, we located a big inverse dating among the capability of bio lm formation and resistance to all antibiotic dealers (p = 0.781), i.e. the bio lm density in touchy traces turned into extra than bio lm density in resistant traces. In constant with us examine, a few researchers additionally proven that the touchy traces tended to supply more potent bio lms than the resistant traces while a few others con rmed that MDR traces had extra functionality for the bio lm manufacturing than touchy traces. (33,34) Bio lm-forming microorganism are embedded in a matrix and gather houses that render them fairly tolerant to antibiotics, UV light, chemical biocides, host immune response, and different outside stresses. Bio lm can defend microorganisms from harsh environmental situations along with hot temperature and pH, excessive salinity and pressure, negative nutrients, antibiotics, etc., with the aid of using appearing as a barrier. Structural barriers, at the side of chronic cells inside bio lm, play a decisive position in antibiotic resistance. As reviews indicate, bio lm-associated infections are tough for remedy and could now no longer be cured easily. Consequently, the prescription of antibiotics will now no longer remedy or take away bio lm-associated contamination because of their antibiotic tolerance and genetic mutation. Bio lm is now taken into consideration to be a number one reason of continual contamination, and antibiotic-resistant microorganism are popular in bio lm form. Currently, it's miles believed that over 80% of continual infectious illnesses are as a result of bio lm, and it's miles recognized that traditional antibiotic medicinal drugs are insu cient at removing those bio lm-mediated infections. (35) In end in our examine improvement of resistance with the aid of using P. aeruginosa to many antimicrobial dealers is a rst rate undertaking in controlling its infections. Therefore, the transmission of those isolates to sufferers ends in better resistance. In total,>50% of P. aeruginosa scienti c isolates had been manufacturers of sturdy bio lm. Therefore, the vital hygiene measurements ought to be taken for the prevention of moving the P. aeruginosa isolates to hospitalized sufferers. The authors report no con icts of interest in this work.