Prevalence and antibiogram of Pseudomonas aeruginosa and Staphylococcus aureus clinical isolates from burns and wounds in Duhok

Introduction: this study aimed to isolate P. aeruginosa and S. aureus , investigate the antimicrobial resistance of collected isolates, and investigate the distribution of exoU and mecA genes in P. aeruginosa and S. aureus isolates. Methodology: Out of 150 samples, 32 isolates were identified as P. aeruginosa , 48 isolates were identified as S. aureus. All isolates were checked for AST. Then, a PCR was applied to detect exoU and mecA genes in P. aeruginosa and S. aureus . Results: 12.0% and 29.3% of the samples showed co-isolates and single isolates of studied pathogens, respectively. Regarding burn samples, S. aureus was the most prevalent pathogen (38.0%, 38/100) among males (41.8%, 23/55), followed by P. aeruginosa (27.0%, 27/100) among females (28.9%, 13/45). The highest burn infection rates of S. aureus (50.0%) and P. aeruginosa (32.7%) were recorded among age groups (≥ 50) and (18-49), respectively. Comparatively, wound samples were less infected with these pathogens. P. aeruginosa isolates usually exhibited high resistance to gentamicin, tobramycin, and netilmicin, whereas, imipenem showed low resistance at 46.87%. S. aureus isolates were susceptible to trimethoprim-sulphamethoxazole and rifampin. 56.25% of P. aeruginosa isolates were exoU positive and 37.5% of S. aureus isolates were mecA positive. Results of the cefoxitin inhibition zone with mecA gene amplification, 33.3% isolates were MRSA, 4.2% isolates were nmrMRSA, and 62.5% isolates were MSSA. Most of the resistant isolates of P. aeruginosa carried the exoU gene, 80% resistant isolates to imipenem were exoU positive. Conclusions: S. aureus was more predominant than P. aeruginosa in burns and wounds infections.


Introduction
The main epithelial barrier between the human body and the hostile environment is the skin which is one of the largest immune organs in the human body [1].A painful traumatic injury of the skin by burns or wounds can cause lowering host immunity, increased hospital prolonging, and ultimately risks of hospital-acquired infections [2].
Pseudomonas aeruginosa (P.aeruginosa) is an opportunistic pathogen responsible for hospitalacquired infections [3], especially bacteremia, and urinary, respiratory, and gastrointestinal tract infections [4], additionally, P. aeruginosa infections can be severe in burnt people, especially with a compromised immune system [5].Abbasi et al., (2017) revealed that this pathogen is the third nosocomial pathogen after S. aureus and Escherichia coli (E.coli) [6].Although P. aeruginosa produces numerous virulence factors, for instance, pili, flagella, proteases, elastase, lipases, iron chelators, and a variety of various toxins, including pyocyanin and exotoxin A [7], The type III secretion system (T3SS) toxins (exoS, exoT, exoU, exoY) were identified as the major virulence determents that transport via T3SS system from pathogen cytosol directly into the cytoplasm of the eukaryotic host cell [8].S. aureus is considered one of the most clinically important zoonotic pathogens, it can cause skin and soft tissue infections, and bloodstream infections [9].
Antimicrobial resistance is one of the most important global threats to general public health [10,11].The spreading of multidrug-resistant (MDR) pathogens reduces the efficacy of antimicrobial drugs, thereby prolonging hospital stays and increasing treatment costs and fatalities.Also, one of the most important pathogens related to antimicrobial resistance worldwide is Staphylococcus aureus (S. aureus) [12].It can resist almost all lactams (e.g., methicillin, oxacillin, and flucloxacillin) and other major antimicrobial classes [13].One of the most common resistance mechanisms in S. aureus is methicillin resistance, which is usually conferred by alteration of penicillinbinding protein-2a (PBP-2a), this protein is located in the bacterial cell wall and has a low affinity for βlactams [14].Historically, methicillin-resistant S. aureus (MRSA) was first reported in 1961 [15].The gold standard for identifying MRSA strains is by detection of the mecA gene which is located on the staphylococcal chromosome cassette mec (SCCmec) in S. aureus [16].
Herein, the objectives of the present study were: (i) to isolate and identify P. aeruginosa and S. aureus from hospitalized patients with burns and wound infections using conventional and molecular methods, (ii) to investigate the antibiogram of collected isolates using Kirby-Bauer disc-diffusion technique, and (iii) to investigate the distribution of virulence and resistance genes of these isolates using species-specific primers.

Study setting
This prospective study was conducted on patients at Burns and Plastic Surgery Hospital, Duhok City, Iraq between October, 2021 and February, 2022.A total of 150 swab samples from admitted patients of different ages and genders having wounds (accident and surgery) or burns were aseptically collected with the help of nurses.The admitted patients did not receive antibiotics previously.For the immediate and post-treatment, all patients received a treatment based on a dressing protocol including disinfection, cleansing, application of topical antibiotic silver sulfadiazine 1% (Awamedica, Iraq), and application of nonadherent gauze, followed by a tibio-breech bandage.Usually, this tropical antibiotic is applied daily.Demographic data collected in this study included sample source, patient age, and gender.

Ethical approval and consent to participate
The PhD proposal with informed consent was approved by the ethics committee of the College of Science, University of Duhok, and Duhok Public Health Directorate, Duhok City, Kurdistan Region-Iraq.Before collecting the clinical swabs, written formal consents for participation were obtained from all eligible patients or parents/guardians of eligible children (Reference number of research ethics was 18082021-8-12 on 7 Jun 2022).

Sample collection and processing
A total of 150 clinical swabs, 100 (55 males: 45 females) burn swabs and 50 (32 males: 18 females) wounds (pus or discharge) swabs were aseptically collected.All swab samples were labelled and immediately transported in ice-packed boxes to the laboratory for bacteriological analysis.The samples were identified as P. aeruginosa and S. aureus by application of selective culture media, differential staining, and different confirmatory tests.The collected swabs were cultured on cetrimide agar (Neogen, USA) and mannitol salt agar (Neogen, USA) under aerobic and sterile conditions and the plates were incubated at 37 °C for 24 hours.After the incubation period, purified colonies on both culture media were selected according to macroscopical characteristics [17,18].Afterwards, the Gram staining technique (Atom, UK) and motility test (slide method) were performed for all purified isolates.Lastly, the obtained results were confirmed using biochemical tests including catalase (slide method) (Scharlau, Spin) and oxidase (using strip) (Bioanalysis, Turkey) tests for confirmatory identification of P. aeruginosa, while catalase (slide method) and coagulase (tube method) tests for confirmatory identification of S. aureus.For further processing, confirmed isolates of both bacterial species were preserved frozen in 2mL eppendorf tubes at -20 °C in nutrient broth supplemented with 25% (v/v) glycerol (Scharlau, Spin) [19].

Extraction of bacterial DNA
Bacterial genomic DNA from P. aeruginosa and S. aureus isolates was extracted using AddPrep Bacterial Genomic DNA Extraction Kit (Addbio, Korea) according to manufacturer's instructions.Then, DNA concentration was measured by NanoDrop spectrophotometer instrument (Thermos Scientific, USA), and DNA purity was measured by reading the 260/280 absorbance ratio [23].Then, extracted DNA from pure cultures was stored frozen at -20 °C.

Molecular detection
In this current study, uniplex polymerase chain reaction (PCR) technique was used for the identification and investigation of virulence and resistance genes of P. aeruginosa and S. aureus using published specific primers for each gene as shown in Table 1.
Each PCR reaction was done in a total volume of 20 µL as follows: 10µL of Add Taq Mater (20mM Tris-HCl (pH 8.8), 100Mm KCl, 0.2%Triton® X-100, 4Mm MgCl2, Protein stabilizer, sediment, loading dye and 0.5 Mm each of dATP, dCTP, dGTP, and dTTP) (Addbio, Korea), 6µL of deionized distilled water, 2µL of DNA template (50 ng/µL) and 1µL of each forward and reverse primer (10 pmol/μL) (Macrogen, Korea).The PCR condition for each studied gene was carried out as shown in Table 2. Following amplification, aliquots of 5µL from each PCR product were analyzed by gel electrophoresis (composed of 1% agarose in TBE buffer (Addbio, Korea)) stained with safe gel stain dye (Addbio, Korea) (5µL/50 mL agarose gel) for 40-45 minutes at 85 voltages.Finally, the PCR products were visualized under a UV transilluminator instrument.The 100bp plus DNA ladder (GeneDireX, Taiwan) was used as the DNA molecular weight standard in this study.

Statistical analysis
Data obtained from this study were primarily input using Microsoft Excel Worksheet 2016 (Microsoft, USA), and descriptive statistics (mean, median, percentage) were calculated.Data analysis was performed using GraphPad Prism 8 (GraphPad Software, USA).Fisher's exact was applied to assess significant associations between two categorical variables.A p value < 0.05 was taken to indicate statistical significance.

Socio-demographic characteristics of patients
A total of 150 participants were enrolled in the current study (63, 42% females and 87, 58% males) with a sex ratio of 0.72: 1.The ages of the participants ranged from 1 year to 85 years with a mean of 28.03 years and a median age of 24 years.Most of the study participants were in the age group of (18-49) years (76, 50.7%).

Bacterial identification
According to the results of morphological, biochemical, and molecular characterization among the 100 burns and 50 wounds swabs, co-isolates of studied pathogens were found in 18 (12.0%)clinical samples, and single isolates were found in 44 (29.3%) samples.Eighty-eight samples (58.7%) showed the absence of studied bacterial pathogens as shown in Figure 1A.S. aureus was the commonest isolated pathogen (48, 60.0%) followed by P. aeruginosa (32, 40.0%) with a significant statistical difference (p value 0.049) as shown in Figure 1B.There were also significant statistical differences between the frequency rate of each pathogen and total clinical samples of burns and wounds as in Table 3 and 4.   Regarding S. aureus infections, burn samples were more infected with this pathogen, especially among males (41.8%), but no significant difference between the pathogen frequency rate and total clinical samples of burns and wounds was noticed (p value 0.252).In females and compared to wounds, burn samples (33.3%) were more infected with this pathogen with a significant statistical difference between the pathogen frequency rate and total clinical samples of burns and wounds (p value 0.025).The highest infection rate (50.0%) was recorded among the age group (≥ 50) of burn patients with a significant statistical difference between the frequency rate of this pathogen in total samples of burns and wounds (p value 0.027).
In P. aeruginosa infections (Table 4), burn samples were more infected with this pathogen especially among females (28.9%) with on significant difference between the pathogen frequency rate and total clinical samples of burns and wounds (p value 0.050).In the male gender and compared to wounds, burn samples (25.5%) were more infected with this pathogen with no significant difference between the pathogen frequency rate and total samples of burns and wounds (p value 0.179).The highest infection rate (32.7%) was recorded among the age group (18-49) of burn patients with significant statistical differences between the frequency rate of this pathogen and total samples of burns and wounds (p value 0.176).However, compared to burns, the occurrence of S. aureus and P. aeruginosa pathogens was less in wounds.

Antimicrobial susceptibility testing
In the current study, most of the P. aeruginosa isolates exhibited higher resistance rates to 10 antimicrobials tested as shown in Table 5. High resistance rates were seen among P. aeruginosa isolates toward aminoglycosides (gentamicin 84.37%, tobramycin 81.25%, and netilmicin 81.25%).Whereas, 53.12% of P. aeruginosa isolates were susceptible to imipenem.S. aureus isolates were usually susceptible to 10 antimicrobials tested, high susceptible rates were seen among S. aureus isolates to trimethoprimsulphamethoxazole (62.50%), and rifampin (60.42%).While, 91.67% and 70.83% of S. aureus isolates were resistant to penicillin G and azithromycin, respectively (Table 6).Before admitting, the patients did not receive any antibiotics.The tested antibiotic, trimethoprimsulphamethoxazole used in the current study and the tropical antibiotic silver sulfadiazine which was used in the immediate and post treatments are both belonging to the sulfonamide or sulfa drug group.

PCR amplification of screened genes
Table 7 summarized the finding of the molecular detection (using conventional PCR) of studied genes in 32 and 48 isolates which were phenotypically identified as P. aeruginosa and S. aureus, respectively.In this current study, 32/32 (100%) and 18/32 (56.25%) of P. aeruginosa isolates were PA-SS positive and exoU positive respectively, while the remaining 14/32 (43.75%) of P. aeruginosa isolates failed to produce the band of 1572 bp specific for exoU gene as shown in Figures 2A and 2C.
Amongst S. aureus isolates, 48/48 (100%) and 18/48 (37.5%) were NUC positive and mecA positive respectively, while 30/48 (62.5%) of S. aureus isolates failed to produce the band of 154 bp specific for mecA gene as shown in Figures 2B and 2D Moreover, S. aureus strains were emphasized by the results of PCR of mecA gene amplification and cefoxitin disc-diffusion methods.However, 16 (33.3%)isolates, 2 (4.2%) isolates and 30 (62.5%) isolates of S. aureus were identified as MRSA, non-multidrug resistant methicillin-resistant Staphylococcus aureus (nmrMRSA), and methicillin-susceptible Staphylococcus aureus (MSSA) strains, respectively as shown in Table 8.Actually, out of 20 isolates of cefoxitin-resistant S. aureus, only 2 isolates failed to produce the band of 154 bp specific for the mecA gene.Whereas all 30 cefoxitin-susceptible S. aureus isolates were mecA-gene negative.
Table 9 illuminated the correlation between antimicrobial susceptibility patterns and possession of exoU gene in P. aeruginosa isolates, both resistant and intermediate-resistant isolates were considered as resistant in this study.The results concluded that most of the resistant isolates to antimicrobial agents tested in this study carried the exoU gene, in particular, 12 (80%) of 15 resistant isolates to imipenem carried the exoU gene with the significant statistical difference between exoU-positive strains and a total number of imipenemresistant and imipenem-susceptible isolates (p value 0.015).

Discussion
Burn infections are the most serious health problem worldwide.Skin damage by burns or wounds leads to exposing a huge portion of its tissue to infectious agents, and this damaged tissue will be a suitable place for residing opportunistic microbes responsible for inflammation and infection immediately after colonization and bacterial secretions [28].The current study investigated the prevalence and susceptibility of P. aeruginosa and S. aureus isolates.The identification of burn/wound-infected patients was based on morphological characteristics, microscopic examination, biochemical tests specific for each isolate, and molecular detection of housekeeping gene specific for each isolate.The results revealed that the prevalence of housekeeping genes PA-SS in P. aeruginosa and NUC in S. aureus isolates was (100%).
S. aureus was frequently co-isolated with opportunistic pathogen P. aeruginosa in polymicrobial infections such as burns and wounds infections [29].Co-infections caused by multiple bacterial species are more virulent and/or more difficult to treat than infections caused by either bacterium alone [30].Although many bacterial species were found in polymicrobial infections, the most common association was between S. aureus and P. aeruginosa [31].
In the current study, S. aureus was found to be the most common pathogen causing burns and wound infections.Globally, S. aureus is an important cause of hospital-acquired and community-acquired infections [32][33][34], due to its ability to produce various virulence factors, including capsule, protein A, a variety of exotoxins and enterotoxins.The prevalence of S. aureus and P. aeruginosa in burn/wound-infected patients was 60.0% and 40.0%, respectively.However, there are arguments about which bacteria is the leading infective pathogen in burns and wounds infections.Our findings were in agreement with previous studies, which have shown that S. aureus was the most prevalent pathogen than P. aeruginosa in burns and wounds [31,35].However, the results were disagreed with the other previous studies by Singh et al. [36] from India, Bayram et al. [37] from Turkey, Anuradha et al. [38] from India, Chaudhary et al. [39] from Pakistan, and Ghafil & Fleih [40] from Iraq, where P. aeruginosa prevalence was more than that of S. aureus.This difference in prevalence rates might be attributed to geographical variations, climatic features, antimicrobials abuse, and tropical antibiotics tested in the immediate and post burn treatments.In this study, more males (41.8%) with burn infections were affected by S. aureus compared to females (33.3%), and higher occurrence of S. aureus among burn patients found in age group (≥ 50) (50.0%) followed by age group (18-49) (42.3%).Regarding P. aeruginosa infections, burns samples were more infected with this pathogen especially among females (28.9%) with the highest burn infection rate (32.7%) recorded among the age group (18-49) followed by age group (≥ 50) (25.0%).These results indicated that burn infections in the elderly were quite more than infections in a younger population due to age-related alternations in immunity.
Most S. aureus isolates were susceptible to antimicrobial drugs tested in this study; high susceptibility rates were observed among S. aureus isolates to trimethoprim-sulphamethoxazole 62.50% followed by rifampin (60.42%).While, 91.67% and 70.83% of S. aureus isolates were extremely resistant to penicillin G and azithromycin, respectively.The reason of high susceptibility of S. aureus to trimethoprimsulphamethoxazole might be using of the antibiotic silver sulfadiazine which was used in the immediate and post treatments.So, it might result in providing selection condition for trimethoprimsulphamethoxazole.These results were consistent with the finding of studies by Bhat & Vasaikar [35], Ansari et al. [41], and Chen et al. [42], which have shown that S. aureus isolates were extremely resistant to penicillin G.However, according to another study conducted by Ahmed et al. [43] from Pakistan, S. aureus isolated from burn wounds were highly resistant to trimethoprim-sulphamethoxazole.
P. aeruginosa has multidrug resistance mechanisms, for instance, it can develop antimicrobial resistance through chromosomal mutations, and the acquisition of resistance genes encoding β-lactamases [44], subsequently, the action of antimicrobial drugs becomes limited.In this current study, P. aeruginosa isolates usually exhibited higher resistance rates to antimicrobial drugs tested.However, P. aeruginosa isolates were highly resistant to gentamicin, followed by tobramycin and netilmicin.Whereas, P. aeruginosa isolates were more susceptible to imipenem antibiotics which relatively revealed that this antibiotic was the last choice of therapy for burns and wounds infections.This was in accordance with the study conducted in Northwest Iran by Azimi et al. [45], where P. aeruginosa isolates have the highest rate of resistance to gentamicin and tobramycin, and with the studies conducted by Bhat & Vasaikar [35] from South Africa, Bayram et al. [37] from Turkey, and Bobai et al. [46] from Nigeria, where P. aeruginosa isolates were more sensitive to imipenem.But according to another study conducted by Chaudhary et al [39] from Pakistan, where P. aeruginosa isolates were less sensitive to imipenem (23.3%).The treatment becomes very difficult in burn infections caused by this pathogen and the mortality rate is likely to reach up to 40-50% [47].However, imipenem and meropenem are used for a long period as the last choice antibiotics for the treatment of multidrug-resistant P. aeruginosa infections when other antimicrobial drugs have failed [48].Nevertheless, this study highlighted P. aeruginosa as a multi-drug resistant pathogen in burns and wounds, this could have the following explanations: the present antibiotics policy in the Burns and Plastic Surgery Hospital -Duhok city has caused an increased rate of P. aeruginosa to commonly used antibiotics; the overuse of available antibiotics which are used as prophylactic or therapeutic measures; and introducing broad spectrum antibiotics such as imipenem that can be helpful in treatment of burn/wound infected patients.
In this current study, conventional PCR amplification was employed for the detection of the methicillin resistance gene (mecA) in all S. aureus isolates by using species-specific primers.In addition to the detection of the mecA gene in this study, the cefoxitin disc-diffusion method was performed to identify MRSA strains, according to the results of cefoxitin inhibition zone with mecA gene amplification, 33.3%, 4.2%, and 62.5% isolates of S. aureus were identified as MRSA, nmrMRSA, and MSSA strains, respectively.The prevalence of MRSA in this study found to be 37.5% which was comparable to that previously found in Pakistan by Junaid et al. [49] and in Northeast Ethiopia by Tsige et al. [50], but incomparable to that previously found in Iran by Emaneini et al. [51] (63.6%) and in Iraq by Aalaa and Abd Al-Abbas, (2019) [52] (93.0%),where S. aureus strains isolated from burns carried the mecA gene.
Cefoxitin incorrectly does not identify only 2 out of 20 isolates as resistant that were mecA negative.This was in accordance with the study conducted by Zhu et al. [53], where only 3 out of 115 cefoxitin-resistant isolates were mecA negative.The explanation for two mecA-negative isolates with cefoxitin-inhibition zone (10-21mm) was maybe regarded with the concentration of antibiotic power in cefoxitin-30μg disc.On the other hand, Ba et al. [54] mentioned specific alterations in different amino acids present in penicillin-binding proteins cascade (PBPs 1, 2, and 3) which may be the basis of resistance.However, a study conducted by Swenson et al. [55], which had shown that the cefoxitin disc-diffusion test is preferred over the oxacillin discdiffusion test for detecting mecA-mediated methicillin (oxacillin) resistance in S. aureus.In addition, mecC designed as the recently reported mecA homologue in the detection of the MRSA strains.Based on our results, we recommend other researches to establish the prevalence of the mecA and mecC genes among phenotypically identified MRSA strains and their effectiveness against different antibiotics in clinical specimens.
Also, conventional PCR amplification was used for the detection of the virulence gene (exoU) in P. aeruginosa isolates and 56.25% of P. aeruginosa isolates were exoU positive.exoU toxin as a cytotoxic protein has phospholipase activity towards the cell membranes of mammalian host cells resulting in rapid and complete cell lysis (cellular necroptosis) [56].However, all P. aeruginosa strains do not have the T3SS toxins, for instance, the exoU gene was found in 28-42% of isolates from acute infections [57].According to the results about the correlation between antimicrobial susceptibility patterns and possession of exoU gene in P. aeruginosa isolates, most of the resistant isolates to antimicrobial agents tested in this study carried exoU gene, in particular, (80%) of resistant isolates to imipenem were carried exoU gene.Our findings are in agreement with previous studies by Subedi et al. [58] and Takata et al. [59], which have shown that most of the carbapenem-and fluoroquinolone-resistant strains of P. aeruginosa were exoU positive.exoU-positive P. aeruginosa strains tend to harbour mutations in quinolone resistancedetermining regions that lead to fluoroquinolone resistance [60].Strains possessing the exoU gene were more resistant to the antibiotics tested in this study.However, other researchers should investigate the presence of other exo (exoS, exoT, and exoY) genes among phenotypically identified P. aeruginosa and their correlations with antibiotic resistance.

Conclusions
It was noticed that S. aureus was more predominant than P. aeruginosa in burns and wounds infections.Burns samples were more infected with S. aureus especially among males.S. aureus isolates were susceptible to trimethoprim-sulphamethoxazole and rifampin, while more resistant to penicillin G and azithromycin.P. aeruginosa isolates exhibited higher resistance rates to antimicrobial tests especially gentamicin, tobramycin, and netilmicin while having a low resistance rate to imipenem.The cefoxitin discdiffusion test was used successfully for detecting mecAmediated methicillin (oxacillin) resistance in S. aureus isolates.Most of the P. aeruginosa isolates exhibit resistance to antimicrobial agents especially imipenem was exoU positive.

Figure 1 .
Figure1.A: Frequency rates of single isolates and co-isolates of studied bacterial pathogens; B: Frequency rates (%) of S. aureus and P. aeruginosa strains in burns and wounds.

Table 1 .
Specific genes, primers sequences, and expected products for PCR assays for amplification of studied genes in P. aeruginosa and S. aureus isolates.

Table 2 .
Conventional PCR programs of PA-SS, NUC, exoU, and mecA genes amplification in collected isolates.

Table 3 .
Proportions (%) of S. aureus isolates among variable genders and ages.

Table 5 .
Percentages of antimicrobial susceptibility rates of isolated P. aeruginosa from burns and wounds.

Table 6 .
Percentages of antimicrobial susceptibility rates of isolated S. aureus from burns and wounds.
†Cefoxitin resistance as a surrogate maker for the detection of methicillin (oxacillin)-resistant Staphylococcus aureus (MRSA) strains.

Table 7 .
Molecular detection of studied genes in (32) P. aeruginosa and (48) S. aureus isolates in this study.

Table 8 .
Comparison of the results by PCR amplification of mecA gene and standard disc-diffusion method of cefoxitin for demonstration of S. aureus isolates strains.

Table 9 .
Antimicrobial susceptibility patterns and possession of exoU gene in P. aeruginosa isolates.