1. Introduction

Urinary tract infections (UTIs) are one of the most prevalent infectious diseases observed in both outpatients and hospitalized patients (Hernandez et al., 2021; Morado and Wong, 2022; Petca et al., 2021). They are a major health problem affecting 150 million people globally each year and one of the most common reasons for adults seeking medical advice (Onanuga & Awhowho, 2012; Shigemura et al., 2005). It is common knowledge that Staphylococcus spp., from Gram-positive bacteria, are the second most common bacteria after Escherichia coli which cause UTI among inpatient and outpatients (Balamurugan et al., 2015; Onanuga & Awhowho, 2012). Generally, Staphylococcus aureus (S. aureus) is a commensal as well as pathogenic bacterium that it did not only cause a wide range of infections in clinical cases (Baraboutis et al., 2010), but also contributed about 13% to cause series bacteremic UTIs in a large community hospital that predominantly affected older patients (Baraboutis et al., 2010). The thermostable nuclease of S. aureus is encoded by the nuc gene, and the PCR for amplification of this gene has the potential to rapid the diagnosis of S. aureus (Brakstad, et al., 1992). Besides, S. aureus that is resistant to methicillin was first identified in the 1950s, and its prevalence has significantly increased over the past several decades (David & Daum, 2010; Grundmann et al., 2006). Studies reported that MRSA strains are a major problem in hospitals, geriatric nursing homes, and other healthcare (Archer & Pennell, 1990; Grundmann et al., 2006; Unal et al., 1994a). According to WHO (WHO, 2021), 64% of infected patients with MRSA are more likely to die than those infected with S. aureus, are sensitive to antibiotics (WHO, 2021).

In 1990, a study noted that the mecA gene was not found in methicillin-susceptible isolates of staphylococci (Archer & Pennell, 1990; Louie et al., 2000a). However, later a study demonstrated that the mecA gene was regarded as the benchmark for identifying methicillin-resistant Staphylococcus aureus (MRSA) (Monsen et al., 2003; Unal et al., 1994b). Further studies reported that MRSA strains bearing the genes encoding for Panton-Valentine leucocidin (PVL), a highly powerful toxin, have been accountable for a serious threat to public health (Holmes et al., 2005; Sina et al., 2018). In addition to ica genes, it is known that the intercellular adhesion (ica) locus genes present in Staphylococcus spp. (Cramton et al., 1999) and the expression of these genes will activate the capsular polysaccharide of S. aureus (Namvar et al., 2013). Gad (Gad et al., 2009) reported that icaA genes play a significant role in biofilm formation in S. aureus. The creation of bacterial biofilm-like communities within the urinary bladder complicates treatment because their adhesion to uroepithelial tissues is crucial for ascending infection (Balamurugan et al., 2015). Besides, these genes are also responsible for protecting the bacteria from the host immune system and antibiotic therapy (Ribeiro et al., 2012).

In the last few decades, S. aureus has emerged as the most prevalent Methicillin-resistant bacterium in the globe and it has become the most often isolated species among Staphylococcus in various clinical samples (Grundmann et al., 2006; Harkins et al., 2017; Louie et al., 2000b). Although studies reported that MRSA is significantly associated with urinary catheterization (Gad et al., 2009; Ibtissem et al., 2013; Muder et al., 2006; Walker et al., 2017) and antibiotic use (Nandhini et al., 2022), recent incidents recorded that MRSA occurs among UTIs out-patients as well (Ahmed et al., 2014; Mitiku et al., 2021b). Since there is not a thorough surveillance program of community-acquired UTIs caused by MSRA in Kurdistan Region-Iraq, this study was undertaken to assess the prevalence and antimicrobial susceptibility pattern of MRSA in Zakho Hospital. It is believed that this is the first article from Kurdistan Region-Iraq that describes the mecA, icaA and PVL of MRSA and causes of community-onset UTIs.

2. Materials and Methods

2.1 Patients and samples collection

This current study was conducted at Zakho General Hospital in Zakho City, Kurdistan Region, Iraq, from August 2021 and March 2022. A total of 402 midstream urine specimens of symptomatic outpatient UTIs (less than 80 years) and they had some symptoms of the following; burning, dysuria, as well as discomfort, pain in the pelvic and back region, and polyuria and confirming that patients did not receive antimicrobials treatment.

2.2 S. aureus isolation and Identification

All urine samples were directly cultured on Mannitol Salt Agar (MSA) and Blood Agar (BA) (5% of sheep blood) and incubated aerobically for 24 hrs. at 37°C. A specimen was considered positive for UTI if a single organism was cultured at a concentration of 10⁴-10⁵ CFU/ml (A. Silva et al., 2022a). Then sub-cultured on Mannitol Salt Agar and incubated aerobically at 37°C for 24hrs. The identification of S. aureus based on the standard microbiological protocols and biochemical characteristics of these pure colonies includes Gram-staining (Atom Scientific Ltd, UK), MSA (Neogen Ltd, UK), and catalase and coagulase tests (Oxoid Ltd, England) (Bale et al., 2021; Selim et al., 2022).

2.3 Antimicrobial susceptibility testing and Oxacillin test

All S. aureus was tested for antibiotic sensitivity patterns using the Kirby-Bauer method (Disc Diffusion Method) (Biemer, 1973; Omar, 2014), including MRSA detection by Oxacillin disc test (Velasco et al., 2005). This was performed on Mueller–Hinton Agar with the following antibiotic discs (Bioanalyse Antimicrobial Susceptibility Testing Discs, Turkey); Imipenem (IPM; 10μg), Rifampin (RA; 5μg), Gentamicin (CN; 10μg), Ciprofloxacin (CIP; 10μg), Amikacin (AK; 10μg), Norfloxacin (NOR; 30μg), Meropenem (MEM; 10μg), Amoxicillin/clavulanic acid (AMC; 20/10μg), Levofloxacin (LEV; 5μg), Cephalexin (CL; 30μg), Oxacillin (OX; 5μg), Trimethoprim (TMP; 10μg), Tetracycline (TE; 10μg), Cloxacillin (CX; 10μg), Cefotaxime (CTX; 30μg), Methicillin (ME; 10μg), Erythromycin (E; 10μg), and Ampicillin (AM; 10μg). The antibiotic discs were then placed on Muller-Hinton Agar and the inhibition zones were measured using a ruler. The sensitivity pattern was scored simply as whether resistant or sensitive according to the Clinical and Laboratory Standards Institute (CLSI, 2007).

2.4 Bacteria DNA extractions

Bacterial DNA was isolated from overnight cultures on nutrient broth at 37°C. Genomic DNA was extracted by using the commercially available kit (Addprep Bacterial Genomic DNA Extraction kit, INC Daejeon, Korea) following the manufacturer’s protocol. The high-quality of extracted bacterial DNA, DNA concentration and purity, was measured by NanoDrop (Thermo Scientific NanoDrop One, United States) and then stored at −20 °C for further investigation.

2.5 Molecular Identification of the specific-species gene of S. aureus and detection of MARSA genes

After phylogenetic identification of isolated S. aureus, all of them were confirmed by PCR amplification using the specific-gene primer (nuc) size (267bp) according to Brakstad (Brakstad et al., 1992) (Table 1 & Figure 1). Then, all S. aureus isolates were tested for the presence of three marker genes of MRSA by Multiplex-PCR amplification of the mecA (310bp), luk-PV (432bp), and icaA (188bp) (Strommenger et al., 2008). Details of the four primer sequences (Macrogen, Seoul, Korea), PCR product sizes and thermocycler conditions are illustrated in Table 1.

2.6 PCR and Multiplex amplification

PCR and Multiplex-PCR was performed using (GeneAmp PCR system 9700 Thermocycler PCR machine). Regarding the nuc gene, the reaction was carried out in a 20µl containing 10µl of 2X Taq PCR Master Mix polymerase (Guangzhou Dongsheng Biotech Co., Ltd.)), 1µl (10 pmol) of each forward and reverse primers and a 2µl of DNA template (100ng/µl), and then added 6µl free nuclease water. In addition, 20µl of the reaction was prepared for the Multiplex PCR, and the tubes of PCR contains 10µl of 2XTaq PCR Master Mix polymerase and 0.5µl for each forward and reverse of three primers, 2µl DNA and then added 5µl of free-nuclease water. The thermocycle condition of PCR amplifications is illustrated in (Table 1).

2.7. Gel electrophoresis for visualization the PCR products

The PCR products for those genes were visualized 1.5% agarose gel in TAE buffer and staining with RedSafe™ Nucleic Acid Staining Solution (20,000x) (iNtRON Biotechnology Co., Ltd. Korea). On the gel electrophoresis, the amplified PCR products were separated (80V, 45mins) and compared to a DNA marker ladder (GeNet Bio, Korea). The gel was exposed to UV light to visualize the bands under UV illumination (Cleaver Scientific Ltd, UK) and expected amplicon sizes are shown in Table (1).

2.8 Statistical analysis

The Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) was carried out online to analyze the distribution of MRSA marker genes among isolates with UTIs. GraphPad Prism version 9.1.4 was used to calculate the Spearman’s correlation coefficient was used for nonparametric correlation between these marker genes of MRSA and antibiotics resistance patterns depending on age with UTIs and the significance was established if p < 0.05.

2.9 Ethical approval

The approval for conducting this study was given by the Ethical Committee of Duhok Directorate General of Health (ethical code n 18082021-8-27) and the Ethical and Protocol Review Committee of the Biological Sciences Committee (BSCZ) at the University of Zakho (ID: “BSCZ/28/7/2021”).

3. Results

3.1. S. aureus isolation with phenotypic and genotypic detection

A total of 402 specimens of (midstream) urine were collected from adult outpatients of both genders with UTI symptoms. The growth bacterial cultures were 293 (72.9%) and from these, 37 (12.6%) were phenotypic identification of S. aureus. Gram-positive cocci, golden yellow colonies on BA and mannitol fermenting yellow color on MSA and positive for both catalase and coagulase tests were done for phenotypic identification. Then, all isolated S. aureus were molecularly confirmed by PCR amplification of the specific-species gene (nuc) and the electrophoresis gel is as shown in Figure 1.

Figure 1. Gel electrophoresis of PCR amplification of the specific-species gene (nuc) of S. aureus isolates

The amplified DNA fragments specific primers nuc gene; lanes for isolated bacteria samples S20-S28 and lane Ladder for 100bp (GDSBio Marker). These amplified DNA fragments were pipetted into a prepared 1.5% agarose gel stained with 5µl of RedSafe™ Nucleic Acid Staining Solution.

3.2 MRSA detection

Isolated S. aureus were subjected to detect whether they are MRSA or not, by Oxacillin Disc test and confirmed by PCR amplification of mecA gene. The amplicon size is 310bp. From these isolated S. aureus, a total of 28 (75.7%) of S. aureus were resistant to Oxacillin and have mecA gene and considered as MRSA. The total number of UTIs with MRSA was 26 (92.9%) and 25 (89.3%) in married and suffered from UTIs chronic cases, respectively. In addition, the prevalence rate of infected females 24 (85.7%) was higher than males. In addition to mecA gene, these isolates MRSA were tested to get both icaA and luk-PV genes. The prevalence rate of both icaA and luk-PV genes was found in 21 (75%) and 18 (64.3%) of isolates MRSA with amplicon sizes 188bp and 432bp, respectively (Figure 1b).

(a)

(b)

Figure 2. Gel electrophoresis for the multiplex PCR assay for detecting mecA, icaA and luk-PV, genes of isolates MRSA (a) and the prevalence rate and total number of these marker genes (b).

The amplified DNA fragments by multiplex PCR assay for the marker genes (a); lane control negative (Control -ve), lane 3-7 for the amplified DNA fragments produced (S22 and S11, possess both mecA and icaA genes, S10 possess both mecA and luk-PV, S20, S19, S18 and S12 have three genes; luk-PV, mecA and icaA genes). These amplified DNA fragments were pipetted into a prepared 1.5% agarose gel stained with 5µl of RedSafe™ Nucleic Acid Staining Solution. The prevalence rate and total number of mecA, icaA and luk-PV genes.

3.3 Distribution and relationship of these marker genes in isolated MRSA

Figure (3a) demonstrates the details of MRSA marker genes harbored in all of the isolated S. aureus samples. A total of 14 (50.0%) of isolated samples possess of mecA, icaA, and luk-PV genes while 7 (25%) of them harbored two genes: mecA, and icaA. Furthermore, 4 (14.3%) of isolated have both genes; mecA and luk-PV, and only 3 (10.7%) have mecA, respectively. In addition, one bacterium has only luk-PV gene. In addition, the Spearman’s correlation coefficient of these data indicated a significant positive correlation between mecA and both virulence marker genes of luk-PV, and icaA in MRSA with UTIs (Figure 3b).

The distribution of MRSA marker genes among isolates with UTIs by Venn Diagram software. A total 14 of MRSA possess three genes; mecA and luk-PV, and icaA and followed by 7, 4, and 3 isolated MRSA possess two genes (mecA, and icaA), and (mecA and luk-PV), and luk-PV, respectively. The non-parametric Spearman correlation analysis indicated that the numbers of MRSA increased with increased both marker genes and the marker denote a sum observation of these genes for a particular age group of UTIs. The significance was considered when p<0.05.

(a)

(b)

Figure 3. The Venn Diagram for the distribution of marker genes among (a) and correlation analysis between the specific-gene (mecA) and both the marker genes of (icaA and luk-PV) (b) of isolates MRSA with UTIs.

3.5 Antimicrobial susceptibility patterns of MRSA

Isolated MRSA were subjected to the antimicrobial susceptibility to determine patterns for the 18 antibiotics disks, and the results are shown in Figure (2).

Isolated MRSA exhibited total resistance to Ampicillin and approximately 96%, 85%, 78%, 78 and 74% for Cefotaxime, Methicillin, Norfloxacin, Cloxacillin, and Amikacin, respectively. Nearly the same percentage 70% was resistant to Trimethoprim, Tetracycline Erythromycin, and Cephalexin. Additionally, around 63% of MRSA isolates were resistant Meropenem and the same percentage of both 56% and 52% were resistant to both Levofloxacin and Gentamicin, and Ciprofloxacin and Rifampin, respectively. By contrast, these isolates MRSA were sensitive to Imipenem 92.6%.

Figure 2. The antibiotics sensitivity patterns of MRSA isolates

Keys: Antibiotic; IPM; Imipenem, RA; Rifampin, CN; Gentamicin, CIP; Ciprofloxacin, AK; Amikacin, NoR; Norfloxacin, MEM; Meropenem, AMC; Amoxicillin/clavulanic acid, LEV; Levofloxacin, CL; Cephalexin, OX; Oxacillin, TMP; Trimethoprim, TE; Tetracycline, CX; Cloxacillin, CTX; Cefotaxime, ME; Methicillin, E; Erythromycin, AM; Ampicillin.

4. Discussion

Uropathogenic bacteria have been identified as a major cause of UTIs, with highly significant morbidity and mortality rates, worldwide (Tula et al., 2016). Generally, Gram-positive bacteria, particularly S. aureus, have emerged as significant contributors to hospital and community-acquired infections and almost are resistant to antibiotics and easily spread (Lunacek et al., 2014). In this study, S. aureus accounted for only 37 (12.6%) of isolates from urine samples submitted from the community. This finding was approximately similar with laboratory-based studies conducted in Iraq; 11.1% in Tikrit City (Al-Jebouri & Mdish, 2013) and 13.5% in Thi-Qar City (Abbas & Hamim, 2019). In addition, this finding was supported by earlier studies that reported that S. aureus was the second most common pathogen in UTIs, and it is more common in women (Onanuga & Awhowho, 2012; Silva et al., 2022a). However, their studies did not show whether S. aureus was MRSA or not.

It is interesting to note that in this study, out of 28 (75.7%) isolates were Oxacillin resistant from the total of 37 clinical isolates of S. aureus, which are considered as MRSA. The incidence of UTIs with MRSA is much higher than what has been reported in recent studies in Iraq; in Baghdad City Khaleel et al. (2021) reported that 7.7% of isolates were positive for MRSA when using Oxacillin and Cefoxitin resistances as a marker for detecting MRSA, In contrast, in Thi-Qar City a study condcuted by Abbas and Hamim (Abbas & Hamim, 2019) reported that all isolated S. aureus were Oxacillin-resistant without mentioning whether they are MRSA or not. Furthermore, the high frequency of MRSA is resembled to those recently found in Khartoum, Sudan (Omar, 2014), where 72% of the detected isolates were MRSA. However, their study methods of identification were different from this study. On the other hand, this finding was much higher than that found in recent studies in South Ethiopia by Mitiku et al., (2021b) and in India by Mendem et al., (2016), lower prevalence of 42.6% and 55.3% among outpatients’ community-acquired UTIs, respectively.

It is emphasized that mecA gene is the specific-genes for the identification of MRSA from clinical samples (Maes et al., 2002; Metri & Jyothi, 2021). In the current study, the genotypic detection of MRSA in UTIs by mecA gene was confirmed for all isolates. Females were highly significant infected than males, particularly among married and chronic UTIs cases. This high frequency of detecting the mecA gene is comparable to this found in Sudan (Ahmed et al., 2014). The incidence of detecting mecA is almost double than that illustrated in a recent study in India (Jyothi & Metri, 2021), with a lower prevalence of 44 % from catheterized patients with UTIs. This variant rate could be explained by identification methods, time and condition of collecting sample and geographical differences (Mitiku et al., 2021b). Indeed, MRSA has become not only a global nosocomial disease and rapid dissemination to healthcare and the community but also it is extremely antibiotic resistant, with variations between institutions and countries (Grundmann et al., 2006; Louie et al., 2000b; Mitiku et al., 2021b). The UTIs with MRSA are probably because of a number of clinical factors, including anatomical variations, hormonal impacts (hormonal changes during pregnancy favor UTIs in females), behavioral tendencies, and physiological causes (Silva et al., 2022b). Because of their smaller urethral length and closer vaginal cavity and rectal entrance (where possible uropathogens reside), females are more likely than males to have germs enter the urethra and climb to the bladder (Silva et al., 2022b). Additionally, it is believed that chronic cases highly sexually active individuals, and child-bearing age groups are the key areas where S. aureus is prevalent (Akortha & Ibadin, 2008; Ramasamy et al., 2019). Other factors associated with urinary tract infection as mentioned above such as gender, age and marriage may play a significant role in contributing to the increased incidence of MRSA in the community. In addition to those factors, the existence of a chronic underlying condition was statistically related with the frequency of MRSA, who has a history of UTIs. This finding is in agreement with a study condcuted in Southern Ethiopia (Mitiku et al., 2021a). They reported that patients with a history of UTI were more likely to have MRSA than those without a history of UTIs. Besides, another reason for the high frequency might be possible MRSA patients who have been discharged from the hospital spreading the infection throughout the community (Lunacek et al., 2014).

In this current study, the high frequency of icaA (75%) and luk-PV (64.3%) in all isolated MRSA, is comparable to a study done in Sudan (Ahmed et al., 2014) where 64% of MRSA possess the icaA gene while it was luk-PV (25%). According to an epidemiological study by Bhatta (Bhatta et al., 2016), PVL genes were linked to increased virulence of community-acquired MRSA from various samples. Thus, the PVL gene has been identified as a reliable marker of community-acquired MRSA strains in various clinical samples (Amin et al., 2020; Holmes et al., 2005; Motamedi et al., 2015). It is noted that a few studies have established antibiotics susceptibility patterns of MRSA with UTIs. In this study, all MRSA was resistant to Ampicillin. This finding was similar to a recent study conducted in Ethiopia (Mitiku et al., 2021b). Furthermore, MRSA was highly resistant to Cefotaxime (92%) and this finding was higher than that found in recent studies (71%) conducted in Sudan (Ahmed et al., 2014), and 74.2% in Egypt (Ibrahim et al., 2020), while MRSA were around 75% resistant to Norfloxacin, and Amikacin and they are nearly close to the finding reported in the recent studies in Ethiopia (Mitiku et al., 2021b) and in Austria (Lunacek et al., 2014). In addition, around 67.8% of MRSA were resistant to Trimethoprim, Tetracycline, Erythromycin, and Cephalexin and these findings were roughly similar to a study done in Iraq (Hamad et al., 2016) and in Khartoum State (Ahmed et al., 2013). By contrast, these isolates MRSA were sensitive to Imipenem 92.8% and this finding was a bit higher than that arrived at in the study conducted in Afghanistan 81.4% (Naimi et al., 2017). The high prevalence resistance rate of MARSA to antibiotics is due to several factors ,namely its ability to form biofilms, by icaA, might be a significant factor in chronic UTIs and antimicrobial drug resistance (Silva et al., 2021; Yousefi et al., 2016). The development of multidrug resistance may be maintained by the slow diffusion of antibiotics through the biofilm matrix, conceivably by selecting highly tolerant strains that are briefly exposed to sub-inhibitory doses of antimicrobial therapy. Di Domenico et al. (2017) reported that the creation of biofilms may give colonizing bacteria important virulence traits ,such as immunity to the host immune system protection and increased general antibiotic tolerance, non-biofilm producers. In addition, The overuse of antibiotics, especially imposes selection pressure on the generation of resistant strains, may potentially contribute to the high prevalence of PVL together with the virulence factor (Amin et al., 2020; Kaur et al., 2012; Motamedi et al., 2015).

This is not suppressing that MRSA strains are typically resistant to multiple antibiotics (Grundmann et al., 2006; Onanuga & Awhowho, 2012; Petca et al., 2021), and it may transmit among people by physical contact and rarely by air according to WHO (WHO, 2021). The role of biofilm formation in these bacteria might also associated with multidrug resistance (Balamurugan et al., 2015). In fact, the community strains' resistance to multiple therapies indicates that they may have originated from the hospital. In addition to these factors, in Iraqi Kurdistan, over the counter and both general practitioners and many nurses have prescribed antibiotics for patients without obtaining the antibiotic sensitivity test from the microbiological laboratory clinic. Therefore, the emergence of MRSA resistant to antibiotics is needed to be well documented and creating strategies for empirical treatment and in assessing the current guidelines (Chambers & DeLeo, 2009).

The main limitation of the study is the lack of sequencing of the 16s rRNA gene of MRSA. Hence studying the phylogenetic tree and whole gene sequencing is essential for understanding the epidemiology and infections in the urinary tract.

5. Conclusions

In conclusion, this study demonstrates that MRSA isolates were the common pathogens from Gram-positive bacteria, particularly married and chronic cases with of UTIs from Zakho City in Iraqi-Kurdistan. Females were highly infected than males and acute cases found in the community-acquired community UTIs. Furthermore, the molecular detections of MRSA strain in UTIs and the highly incidence of infections was strongly associated with both icaA and luk-PV genes as well as multidrug-resistant which were positively associated with these genes. In addition, the most effective antibiotic for treating UTIs, especially with MRSA, is considered to be Imipenem. The recommendations for the use of antibiotics should be monitored by the public health sectors. Further studies of UTIs with S. aureus should investigate whether they are MRSA or not in other cities in Kurdistan Regional-Iraq.

Acknowledgements

The authors are thankful to the Zakho General Teaching Hospital, Department of Biology, Faculty of Science, University of Zakho, and Zakho Technical Institute, for providing some of the research facilities.

Conflicts of Interest

The authors declare no conflict of interest.

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Genes	Primer Sequence (5'_3')	Product size (bp)	PCR Condition for all genes		References
nuc-F nuc-R	5'-GCGATTGATGGTGATACGGTI-3' 5’-AGCCAAGCCTTGACGAACTAAAGC-3'	267bp	Initial denaturation= 95°C for 5 mins,		(Brakstad et al., 1992)
mecA-F mecA-R	5′-GTA GAA ATG ACT GAA CGT CCG ATA A-3’ 5′-CCA ATT CCA CAT TGT TTC GGT CTAA-3'	310 bp	Denaturation= 94°C for 35 Sec. Annealing= 57°C for 90 Sec. Extension= 72°C for 60 Sec.	35 Cycles	(Strommenger et al., 2008)
luk-PV-F luk-PV-R	5'-ATC ATT AGGTAA AAT GTC TGG ACA TGA TCC A-3' 5'-GCA TCA AGT GTA TTG GAT AGC AAA AGC -3'	432 bp		35 Cycles
icaA-F icaA-R	5'-CGAGAAAAAGAATATGGCTG-3' 5'-ACCATGTTGCGTAACCACCT-3'	188pb	Final extension= 68 °C for 10 mins.