The prevalence of mupirocin resistance-producing wound isolates of Staphylococcus aureus

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1 Australian Journal of Pharmaceutical Biology Volume 1; Issue 1; April-June 2018 Original Article The prevalence of mupirocin resistance-producing wound isolates of Staphylococcus aureus Nagaraj Rathika 1, Gnanasekeran Karthikeyan 1,2, Ashokapuram Selvam Saranya 3, Natesan Sudhakar 1,2 * 1 Department of Microbiology, Muthayammal College for Arts and Science, Rasipuram, Namakkal, Tamil Nadu, India, 2 Muthayammal Centre for Advanced Research, Muthayammal College for Arts and Science, Rasipuram, Namakkal, Tamil Nadu, India, 3 Department of Microbiology, Vivekananda College Of Arts and Science for Women, Elayampalayam, Tiruchencode, Namakkal, Tamil Nadu, India ABSTRACT Staphylococcus aureus is one of the predominant nosocomial pathogens which causes a wide range of diseases and post-operative infections. Coagulase-negative Staphylococcus is frequently reported in nosocomial bloodstream infections. It is also associated with implanted medical devices. Mupirocin (MUP) is as an antimicrobial agent. The aim of the present work is to investigate the prevalence of low-level and high-level MUP resistance S. aureus isolates from different wound samples. Three types of samples, skin infection, burn, and accident wound samples, were collected. Out of 45 samples, 18 isolates are accident samples that had highest prevalence of S. aureus and the second highest prevalence of S. aureus was observed in skin infection samples. Among the 18 isolates, 14 (77.7%) of S. aureus were coagulase negative. The highest coagulase negative result was observed in skin infected sample (83.3%), accident and burn infection 77.7% and 66.6%, respectively. Among the 18 isolates, 14 isolates produce low-level MUP-resistant gene. In high-level resistance isolates, the remaining 4 isolates produce high-level MUP resistant gene and were observed by polymerase chain reaction amplification of MUP-resistant genetic and plasmid DNA. Keywords: Coagulase-negative Staphylococcus aureus, multidrug resistant, mupirocin, nasocomial infection Submitted: 16 April 2018, Accepted: 24 June 2018, Published: 30 June 2018 INTRODUCTION Skin is the outer covering which protects us from the environment. But when damaged or opened by means of trauma and wounds, it is constantly exposed to bacteria and hence preventing infections becomes a great challenge. When the skin gets damaged, the bacteria may harbor and multiply to most dangerous levels, which delays the healing activity, which risks in developing bacteremia and septicemia and hence wound infections need to be treated quickly to avoid the consequences (Sukumaran and Senanayake, 2016). Wounds contain a high bacterial prevalence and even if they tend to heal normally, a limited amount of pathogenic bacteria will be present in it. Consequently, when the bacterial count increases, the wound may become infected again that requires antibiotic treatment. If the wound is not healing, it may be a sign of infection. In the wound, the following symptoms indicate the development of infection; odor, elevated exudate level, absent or abnormal granulation, tissue increased pain. The most common causative organisms associated with wound infections include Staphylococcus aureus/methicillin-resistant S. aureus [MRSA], Streptococcus pyogenes, Enterococci, and Pseudomonas aeruginosa (Klein et al., 2007). The emergence of antibiotic-resistant microorganisms and their spread is a life-threatening risk in the medical community. S. aureus is the most common cause of nosocomial infection. Its tendency to multiple antibiotic resistances which often complicates treatments tend the researchers to study more about antibiotic-resistant S. aureus. Special interest in S. aureus surgical site infections is mainly due to its predominant role Address for correspondence:dr. Natesan Sudhakar, Muthayammal Centre for Advanced Research, Muthayammal College for Arts and Science, Rasipuram , Tamil Nadu, India. Phone: / rdcell@muthayammal.in Available at 36

2 in hospital-associated infections and emergence of MRSA (Gosbell et al., 2001). Gosbell vancomycin and linezolid are commonly used antibiotics for MRSA infections whereas mupirocin (MUP), a topical antibiotic, is used for treatment of skin- and soft-tissue infections as well as decolonization of carriers. Many isolates of S. aureus have been found to be resistant to new semisynthetic beta-lactams (methicillin, oxacillin, and flucloxacillin) which become known as MRSA (Archer, 1998; Bania et al., 2006; Jahoda et al., 2007). Infection control programs implement measures to contain the dissemination of MRSA which include efforts to eradicate carriage of S. aureus (Archer, 1998). The MUP antibiotic possesses potent activity against staphylococci and is used against the post-operative wound infections, and also to prevent the nasal carriage of MRSA. However, the widespread use of MUP led to resistance in S. aureus, which has been reported worldwide (Jones et al., 2003). MUP is a topical antibiotic for MRSA decolonization in hospital settings and nursing homes and is used as a highly effective antibiotic against MRSA (Bennett et al., 2014). MUP (pseudomonic acid a) is an analog of isoleucine; it competitively binds to isoleucyl-trna synthetase (IRS), inhibiting protein synthesis. Inhibition of IRS is irreversible and time-dependent, suggesting that the MUP IRS complex is highly stable. The increased use of this antibiotic in individuals colonized with MRSA has been followed by outbreaks of MRSA resistance to MUP (Schmitz et al., 1998). Coagulase-negative staphylococci (CoNS) had been considered to be one of the harmless skin commensals before the 1970s; however, currently has been recognized as important causes of human infections. The CoNS has been recognized as major nosocomial pathogens in the context of prosthetic and indwelling device-related infections. CoNS are also among the most frequently isolated bacteria in clinical microbiology laboratories (Hosseini et al., 2016; Chakraborty et al., 2011). More importantly, CoNS often serve as reservoirs of antimicrobial resistance determinants since they usually have a high prevalence of multidrug resistance. Therefore, it is important to characterize and distinguish S. aureus strains and CoNS (Zhang et al., 2004). Both S. aureus and CoNS share a remarkable ability to accumulate additional antibiotic resistance determinants, resulting in the formation of multidrug-resistant strains. This resistance limits therapeutic options for treatment and substantially increases patient morbidity and mortality. This worldwide resistance problem has made it crucial for clinical laboratories to implement a rapid, accurate, and simple method for the identification and discrimination of MRSA, MUP-resistant S. aureus, and CoNS. Polymerase chain reaction (PCR) experiment has been performed for detection of methicillin and MUP resistance and simultaneous discrimination of S. aureus from CoNS (Abdulmula et al., 2006). The use of PCR for the detection of MUP resistance gene and also determinate the high-level and low-level resistant isolates by plasmid curing. The aim of the present work is to investigate the prevalence of low-level and high-level MUP resistance S. aureus isolate from different wound sample. MATERIALS AND METHODS Collection of Samples Purulent materials were collected aseptically with the aid of sterile swab sticks from 45 patients with different wounds infection at Namakkal District surrounding hospitals. The samples were properly labeled indicating the source, date, time of collection, sex, the age of patients, and the samples were transported in cooler boxes to our Microbiology Laboratory, for bacteriological investigations within 4 6 h of collection. Bacterial Isolation and Identification Culture plates of eosin nutrient agar and mannitol salt agar were used. The swab sticks used for the collection of the samples were streaked directly on the labeled agar plates and incubated at 37 C for 24 h. After incubation, cultures were examined for significant growth. Subcultures were then made into plates of Nutrient agar and incubated for another 24 h. The primary identification of the bacterial isolates was made based on colonial appearance and pigmentation. Biochemical tests were performed to identify microbes. Biochemical tests applied were standard catalase test, citrate utilization, coagulase, oxidase, methyl red, Voges Proskauer, indole production, motility, glucose, sucrose, maltose, lactose, characterization, and identification of the isolates was done using the methods of Cheesbrough, (2006). Coagulase Tube Test The tube is filled with 0.5 ml of 1 in 10 diluted rabbit plasma. To the tube, 0.1 ml of overnight broth culture of test bacteria is added. All the tubes are incubated at 37 C and observed up to 4 h. Positive result is indicated by gelling of the plasma, which remains in place even after inverting the tube (Leski et al., 2016). Antibacterial Stability Test The standard Kirby-Bauer disc diffusion method was used to determine the antimicrobial profiles of the isolates for five antimicrobial agents. Erythromycin, ciprofloxacin, tetracycline, methicillin, and vancomycin. The nutrient broth was prepared and sterilized at 121 C at 15 min and inoculated the isolates then incubated at 37 C for 24 hrs. After incubation period, the broth culture was swabbed into surface of the Mueller-Hinton agar plates, and antibiotic discs were placed, then plates were incubated at 37 C for 18 to 20 h. The zone of inhibition and Available at 37

3 resistance was measured, recorded, and interpreted according to the recommendation of the disc manufacture s standard chart (Bauer et al., 1966). Determination Low-Level Resistance Isolation of genomic DNA 1.5 ml of overnight broth culture was taken in 2 ml microcentrifuge tubes. The tubes were centrifuged at 8000 rpm for 5 min. After centrifugation, the supernatant was discarded and the pellet was collected. The pellet was suspended in 200 μl of 1 TE buffer μl of 10% SDS and mixed by vortexing. The tubes were kept in water bath at 60 C for 20 min. Then, it was added with 300 μl of phenol:chloroform:isoamyl alcohol mixture (24:25:1) to extract the DNA and mixed completely by vortexing. The tubes were then centrifuged at rpm for 10 min to separate the phases. The aqueous phase containing the DNA was carefully removed and transferred to new tubes. Equal volume of 100% isopropanol was added to the tubes containing the aqueous phase. It was mixed by inverting the tubes 3 4 times. The tubes were then centrifuged at rpm for 10 min to pellet the DNA. The supernatant was discarded and the pellet was collected. To the pellet, 200 μl of 70% ethanol was added and centrifuged at rpm for 10 min. Then, ethanol was decanted completely and the pellet was air-dried to give purified DNA. Resuspended the purified DNA pellet in 20 μl of TE buffer. DNA solutions were stored at 4 C for further work. Confirmation of DNA by Agarose Gel Electrophoresis Agarose gel electrophoresis is carried out in a horizontal submarine electrophoresis unit. Thirty ml of 1% agarose gel was prepared with 1 TBE buffer (do not mix) and heated the content to get up to clear solution for casting agarose gel. After cooling the solution, 7 µl of staining dye solution was added into the casting system. The gel was allowed to solidify, and then carefully disassembled from the casting system without disturbing the wells and placed in 1 TBE buffer-filled electrophoresis tank (the buffer level should be above gel). About 5 µl of genomic sample DNA mixed with 2 µl of gel loading dye and then loaded to gel and simultaneously loaded 3 µl of DNA marker provided in the nearby well. The power card terminals were connected at respective positions, run the gel at 50 V until the gel loading dye migrate more than half the length of the gel. Then, switched off the unit and visualized the isolated DNA under UV Transilluminator. PCR All isolates of S. aureus were subjected to PCR assay according to Abdulmula et al. procedure with some modification. The primers were obtained from Sigma, India, and used in the PCR comprised Primer 1 MupA (5 -TAT ATT ATG CGA TGG AAG GTT GG-3 ) MupB (5 -AAT AAA ATC AGC TGG AAA GTG TTG-3 ) for amplification of a 456 bp fragment. Each PCR reaction mixture (20 μl) contained 1 μl of template DNA (Genomic DNA), 2 μl of 10 PCR buffer, 0.5 μl of 2.0 mm of each primers, 1 μl of 25 mm of each deoxynucleotide triphosphate, 0.5 μl of Taq DNA polymerase (Con. 5 U/μl), and 15.5 μl of molecular grade water. The amplification reaction involved an initial denaturation phase at 94 C for 5 min, followed by 10 amplification cycles (denaturation at 94 C for 30 s, annealing at 64 C for 30 s, and elongation at 72 C for 45 s), then 25 further amplification cycles (94 C for 45 s, 50 C for 45 s, and 72 C for 1 min) and a final elongation phase at 72 C for 10 min. After the reaction, 5 μl of the final product was resolved into amplified fragments by electrophoresis in 2% agarose gel at 100 V (45 ma) for 1 h. To estimate the molecular weights of fragments, a 100-bp molecular weight ladder was run on each gel (Ferreira et al., 2011). Determination of High-Level MUP-Resistance by Plasmid Curing Loss of resistance and plasmid was investigated by growing the organisms at 43 C for overnight and screening single colony for the loss of resistance by a replica plate method. Colonies that lost antimicrobial resistance were screened by PCR. This procedure was used to determine the plasmid-mediated antibiotic resistance. Isolation of Plasmid DNA 1.5 ml of overnight broth culture was taken into 2 ml microcentrifuge tubes. The tubes were centrifuged at 10,000 rpm for 10 min. After centrifugation, the supernatant was discarded and the pellet was collected. The pellet was suspended in 100 μl of Solution A and mix the content by vortexing. After vortexing, add 100 µl of Solution B mix the tube by inverting the tube. Add 100 μl of solution C & mix the tube by inverting. Incubate the tubes in room temperature for 30 minutes and centrifuged at 12,000 rpm for 30 minutes. After centrifugation, transfer the supernatant to another tube; add 150 μl of Solution D. Centrifuge the tube at 12,000 rpm for 30 minutes. After centrifugation, the supernatant was discarded and the pellet was collected. In the pellet, 500 μl of absolute alcohol was added and centrifuged at 10,000 rpm for 20 minutes. The supernatant was discarded and the pellet was collected. The pellet was air-dried. Then, add 20 µl TE buffer and stored in the freezer. The plasmid was stored at 4 C for further work (Ferreira et al., 2011). RESULTS Isolation of S. aureus In the present study, 45 patients with different types of wound swab samples were collected during this study period. From the 45 wound swabs, 18 (40%) isolates of S. aureus were isolated by selective culture medium and standard biochemical test [Tables 1-3, Figures 1a-d and 2]. There was a distribution of isolates from the different types of wound infection, accident wound infection was 16 and yielded Available at 38

4 9 isolates (56.2%), skin infection wound swabs were 16 and yielded 6 isolates (37.5%), and burn wound infections were 12 and yielded 3 (25%) isolates of S. aureus. In this study, our prevalence results were subjected to Chi-square analysis. Our results were non-significant to each other s such as age, gender, and types of wound, the results were summarized in Table 4. a c e b Figure 1: Isolation and identification of mupirocinresistant Staphylococcus aureus. (a) S. aureus in MSA plate. (b) Biochemical tests. (c) Catalase test. (d and e) Oxidase and coagulase test. (f) Antibiotic sensitivity test of S. aureus d f Coagulase Test All isolates of S. aureus were performed to coagulase test with human plasma. Among them, 18 isolates 14 (77.7%) of S. aureus showed coagulase negative. The highest coagulase negative result was observed in skin-infected sample (83.3%), accident and burn infection 77.7% and 66.6%, respectively [Table 4 and Plate 1]. Totally 77.1% of antibiotic resistance was observed from coagulase-negative isolates. This study was agreed with other records (Zhang et al., 2004; Hulya et al., 2006; Turutoglu et al., 2006). CoNS had been regarded as harmless skin commensals before the 1970s; however, they are now recognized as important causes of human infections [Table 5, Figures 1d and 3]. Antibiotic Sensitivity Test All the confirmed S. aureus strains were subsequently tested for antibacterial drug resistance based on Kirby-Bauer disc diffusion method. The drug resistance patterns of S. aureus isolated from wound samples were found to be highly variable. Almost all 10 isolates were resistant to one or more antibiotics [Table 2 and Plate 1]. Among the 5 types of antibiotics, the S. aureus isolates show the highest resistance against to vancomycin (80%) and the second most methicillin (100%). The lowest most resistant was against ciprofloxacin (48%). Among the 18 isolates, 5 isolates show highest resistance to all 5 types of antibiotics; the other six isolates show 80% resistance; whereas 4 isolates show 60% resistance and the remaining 2 isolates shows the lowest activity against the tested antibiotics. In this study, totally 9 types of resistance patterns were observed. The result was tabulated in the Tables 5 and 6 and Figure 1e. In recent years, many isolates of S. aureus have evolved resistance to both synthetic and traditional antimicrobial chemotherapy and their prevalence outside the hospital is of potential epidemiological threat (Daum et al., 2002; Kaplan, 2005). Among the 5 types antibiotics, the highest resistance against to methicillin (100%). This result was highest resistance patterns compared to a previous study of Rajaduraipandi (2006), who was observed 33.6% of MRSA isolated from wound pus samples. Figure 2: Occurrence of Staphylococcus aureus from different wound samples Vancomycin is the main antimicrobial agent available to treat serious infections with MRSA, but unfortunately, decrease Table 1: Prevalence of S. aureus from wound samples Sample Age group Total (%) and above M F M F M F M F Wound 1/1 2/2 7/19 3/8 3/9 1/3 0/1 1/1 40.9% Total percentage 100% 37% 33.3% 50% S. aureus: Staphylococcus aureus Available at 39

5 in vancomycin susceptibility of S. aureus and isolation of vancomycin intermediate and resistant S. aureus have recently been reported from many countries. Many reports from North India also recorded the emergence of low level and intermediate vancomycin resistance (Thati et al., 2011) In this study, 80% of vancomycin resistance was observed from our wound isolates. This current record was not correlated to previous results and Hare Krishna, 2009 observed 100% of susceptibility strains of S. aureus. In this investigation, other antibiotic resistance result was followed tetracycline (77%), erythromycin (61%), and ciprofloxacin (48%). Compared to previous studies of Hare Krishna (2009), our result was highest resistance percentage for ciprofloxacin who was observed 26.9% of ciprofloxacin resistance isolates from wound samples (Tiwari and Sen, 2006) [Tables 6-8 and Figure 4]. Identification of low-level MUP Resistance gene from Genomic DNA In this PCR assay, all isolates of S. aureus was subjected according to the previous study. Among the 18 isolates, 14 isolates produce MUP-resistant gene. The distribution of resistant gene from skin infection isolates has highest resistant activity 83.3% and 77.7% from accident wound infection. The burn samples showed the lowest resistance of 66.6%. The mupa gene was not detected in the genomic DNA of any of the low-level MUP resistant isolates, Similarly, Schmitz et al., 2000 did not detect the mupa gene in low-level MUPresistant isolates that they studied (Schmitz et al., 2000) Therefore, more studies are needed to establish whether the observed chromosomal mupa widespread in staphylococci from different geographical regions or whether it is due to the transposition of the mupa determinant or the integration of a MUP resistance plasmids into the bacterial chromosome in those isolates (Mary et al., 1996) [Figures 5 and 6]. Identification of High-Level MUP Resistance Gene from Plasmid DNA In this parameter, isolates were selected based on the results of low-level MUP resistance. In low-level resistance analysis, 4 isolates were not showing the result in PCR. So those isolates were selected for plasmid separation for determinate high-level resistance were present or not. Fortunately, all isolates harboring the high-level resistance that determinate by PCR. This result Table 2: Occurrence of S. aureus from different wound samples Sample source No. of samples % Accident wound Skin Burn infection S. aureus: Staphylococcus aureus Figure 3: Coagulase activity of Staphylococcus aureus Figure 4: Antibiotic resistance patterns for Staphylococcus aureus Table 3: Biochemical character of S. aureus Name of the biochemical test Result Glucose fermentation A + Sucrose fermentation A + Lactose A + Maltose A + Mannitol A + Triple sugar iron A/A Indole production test Methyl red test + Voges Proskauer test Citrate utilization test Catalase test + Oxidase test S. aureus: Staphylococcus aureus Available at 40

6 Table 4: Chi square test Variables Prevalence of Staphylococcus aureus infection Total P values (0.05) χ 2 values Positive isolates (%) Negative isolates (%) Age (100) (37) 16 (59.2) (33.3) 19 (69.2) 12 Above60 2 (50) 2 Gender Male 12 (41.5) 17 (58.6) Female 8 (50) 8 (50) 17 Wound Accident 6 (35.2) 8 (64.7) Skin 6 (46.1) 7 (53.8) 13 Burn 9 (60) 6 (40) 15 Table 5: Coagulase activity of Staphylococcus aureus Total no. of isolates Coagulase positive (%) Coagulase negative (%)% 18 4 (22.2) 14 (77.3) Table 6: Antibiotic resistance activity of Staphylococcus aureus isolates Sample M E CIP T VA % AS1M I I I I R 20% AS2F R R R R R 100% AS3F R S S R R 60% AS4M I S S R S 40% AS5M R R R R R 100% AS6M R R I I R 60% AS7M R I R R R 80% AS11M R R R S R 80% AS16F R R R R R 100% SS17F R R R R R 100% SS18F R I R R R 80% SS21M R R S R R 80% SS22M R R S R R 80% SS27M R R R R R 100% SS29M R I I S R 40% BS33M R I R R R 60% BS35F R R S R R 80% BS44F I R S R R 60% was confirmed by plasmid curing analysis. After curing, the plasmid was subjected to PCR analysis, no amplification bands were observed. From the overall result, the high level of MUP resistance was plasmid-mediated [Table 9 and Figure 7]. Table 7: Standard antibiotic resistance patterns for S. aureus Types of antibiotics % of resistance Methicillin 100 Vancomycin 83.3 Ciprofloxacin 48 Erythromycin 61 Tetracyclin 77.7 S. aureus: Staphylococcus aureus Table 8: Antibiogram pattern identification Antibiotics Total (%) M, V 1 (5.5) M, T 1 (5.5) M, E, T 1 (5.5) M, T, VA 1 (5.5) E, T, VA 1 (5.5) M, CIP, T, VA 2 (11.1) M, E, CIP, VA 3 (16.6) M, E, T, VA 3 (16.6) M, E, CIP, T, VA 5 (27.7) CONCLUSION MUP is a topical antimicrobial agent which is used for the treatment of skin and post-operative wound infections and the prevention of nasal carriage of MRSA. However, the prevalence of MUP resistance in S. aureus, particularly in MRSA, has increased with the extensive and widespread use of this agent in hospital settings. This study characterized low- and high-level MUP-resistant S. aureus isolates obtained Available at 41

7 Figure 5: Comparision of coagulase-negative staphylococci and mupirocin-resistant Staphylococcus aureus Figure 7: Amplification of high-level mupirocin (MUP) gene from plasmid DNA. Lane L4 - MUP gene amplification from non-curing plasmid, Lane MUP gene amplification from curing plasmid, Lane M 1000 bp DNA ladder Figure 6: Amplification of low-level mupirocin gene from genomic DNA Lane 1 - ASIM, Lane 2 - AS2F, Lane 3 - AS3F, Lane 4 - AS4M, Lane 5 - AS5M, Lane 6 - AS6M, Lane 7 - AS7M, Lane 8 - AS11M, Lane 9 - ASl6F, Lane 10 - SS17F, Lane 11 - SS18F, Lane 12 - SS2lM, Lane 13 - SS22M, Lane 14 - SS27M, Lane 15 - SS29M, Lane 16 - BS33M, Lane 17 - BS35F, Lane 18 - BS44F, Lane M bp DNA ladder from Namakkal, Tamilnadu area. MUP-resistant strains are divided into two groups: Low- and high-level resistance. Lowlevel MUP resistance appears to be more prevalent in clinical isolates than high-level resistance. MUP resistance is relatively unusual in S aureus, but it is common and increasing in CoNS. In the period in Greece, a rising incidence of MUP-resistant CoNS has been observed the use of MUP is limited and it is only used for controlling the spread of MRSA. The low-rate of MUP-resistant S. aureus is due to the limited MRSA exposure to MUP and any subsequent development of resistance. High-level resistance to MUP has been associated with the presence of the mupa gene Table 9: Mupirocin resistance activity of Staphylococcus aureus Isolate name Mupirocin LLR Mupirocin HLR AS1M + AS2F + AS3F + AS4M + AS5M + AS6M + AS7M + AS11M + AS16F + SS17F + SS18F + SS21M + SS22M + SS27M + SS29M + BS33M + BS35F + BS44F + found in a plasmid (El-Ghodban et al., 2006). Until recently, the mupa genes encoding high-level MUP resistance was found only on plasmids. Its detection in genomic DNA of isolates expressing low-level MUP resistance (Cheesbrough, 2006). The presence of the determinants for high- and low-level MUP resistance in the same isolate (IBN287) further raised the Available at 42

8 possibility that the mupa determinate may be a transposable element. (Leski et al. (2016) have reported similar finding in Staphylococcus epidermidis. The mupa gene was not detected in the genomic DNA of any of the low-level MUP-resistant isolates. Similarly, Schmitz et al. did not detect the mupa gene in low-level MUP-resistant isolates that they studied. Therefore, more studies are needed to establish whether the observed chromosomal mupa widespread in staphylococci from different geographical regions or whether it is due to the transposition of the mupa determinant or the integration of a MUP resistance plasmids into the bacterial chromosome in those isolates. REFERENCES Archer, G. L. (1998). Staphylococcus aureus: A well-armed pathogen. Clinical Infectious Diseases, 26, Bania, J., Dąbrowska, A., Różalska, B., Sadowska, B., Więckowska- Szakiel, M., Korzekwa, K., Żarczyńska, A., Bystroń, J., Chrzanowska, J., & Molenda, J. (2006). 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