Methicillin-resistant Staphylococcus aureus in urinary tract infections; prevalence and antimicrobial resistance

1Department of Pharmacology, College of Medicine, University of Al Iraqia, Baghdad, lraq 2Department of Pharmacology, College of Medicine, University of Kerbala, Kerbala, Iraq 3Department of General Surgery, Al-Yarmouk Teaching Hospital, Baghdad, Iraq 4Department of Chemistry, Faculty of Science, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 5Department of Internal Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran


Introduction
Urinary tract infections (UTIs) are amid the most critical infections kinds globally. UTIs include varieties of disorders, such as urethritis, cystitis, and pyelonephritis. Reports showed that 50% of women had a history of UTIs in their lives. UTIs are thoughtful health issues that concluded 150 million individuals globally yearly (1).
Reports showed that bacteria are the most common cause of UTIs. However, the Staphylococcus aureus is not documented as a major pathogen responsible for the occurrence of UTIs, but its prevalence has been increased in recent investigations (2). Staphylococcus aureus is a significant human pathogen responsible for most cases of nosocomial and hospitalacquired infections. It is responsible for the occurrence of several diseases, including UTIs, respiratory and Introduction: The newly-launched strain of the Staphylococcus aureus, methicillin-resistant S. aureus, is considered the most emerging bacterium in-hospital infections globally. Objectives: The current research focused on the prevalence and virulence features of methicillin-resistant S. aureus (MRSA) bacteria recovered from urinary tract infections (UTIs) cases. Patients and Methods: A total of 710 urine specimens were taken from hospitalized patients who suffered from UTIs. S. aureus was recovered from urine specimens using the microbial culture. S. aureus antimicrobial susceptibility was assessed toward oxacillin and cefoxitin antimicrobial disk to determine the MRSA strains. The polymerase chain reaction (PCR) assessed the distribution of antimicrobial resistance encoding genes. S. aureus antimicrobial resistance was evaluated by disk diffusion. Results: Fifty-five out of 710 (7.7%) urine specimens were positive for the MRSA bacteria. The uppermost antibiotic resistance was obtained against penicillin (100%), ceftaroline (100%), gentamicin (87.2%), erythromycin (76.3%), and ciprofloxacin (69.0%). BlaZ (100%) and tetK (85.4%) had the higher frequency amid examined antimicrobial resistance-encoding genes. Conclusion: The high prevalence of MRSA isolates harboring antimicrobial resistanceencoding genes in the UTIs suggests that diseases caused by them need more expansion healthcare monitoring with essential demand for novel antimicrobials. soft tissue infections, endocarditis, osteomyelitis, and endocarditis (3). The bacterium has an emergence of severe antimicrobial resistance. Clinical experiences showed that around 50% of the S. aureus isolates harbored complete resistance toward penicillins and cephalosporins groups of antimicrobials (4), which called them methicillin-resistant S. aureus (MRSA). MRSA strains caused complicated diseases for a more extended period with a higher economic burden due to hospitalization and treatment (5).
Some genes are involved in the occurrence of antimicrobial resistance amongst the MRSA strains. High distribution of the genes encodes resistance against penicillins (blaZ), tetracyclines (tetK), macrolides (msrA and ermA), and fluoroquinolones (gyrA) was described in the MRSA isolates of clinical infections (6).
Most UTIs caused by MRSA are healthcare-associated-MRSA (HA-MRSA) infections. Commonly, HA-MRSA's UTI cases are asymptomatic, but symptomatic cases require treatments. However, MRSA strains exhibited whole resistance to all kinds of penicillins and cephalosporins and high resistance toward other antimicrobials types (7).

Objectives
According to the uncertain role of MRSA in UTIs, an existing survey was conducted to evaluate the prevalence and antimicrobial resistance of MRSA bacteria recovered from cases of UTIs.

Urine specimens
From January to November 2020, 710 urine specimens were taken from patients referred to the Al-Yarmouk teaching hospitals, Baghdad, Iraq. Patients were hospitalized owing to severe UTIs. Midstream urine was taken through sterile conditions to reduce possible microbial and artifactual contaminations. Urine specimens were taken using sterile glass tubes (10 mL) and immediately transported to the laboratory at 4°C.

DNA extraction and quality assessment
Tryptic Soy Broth (TSB) (Merck, Germany) was used for MRSA growth before DNA extraction. DNA extraction kit (Thermo Fisher Scientific, Germany) was applied. The NanoDrop (NanoDrop, Thermo Scientific, USA) device was applied for the quantitative assessment of extracted DNA. Agarose gel electrophoresis (2%) was applied to the qualitative assessment of extracted DNA. Table 1 displays the polymerase chain reaction (PCR) circumstances applied for this goal (10,11). Eppendorf Mastercycler (Hamburg, Germany) device was applied for the amplification. Positive (positive DNA samples of each gene) and negative [PCR-grade water (Thermo Fisher Scientific, Germany)] controls were applied to monitor the findings of the PCR.

Data analysis
Data collected from the experiment were numerically evaluated by the SPSS/22.0. Qualitative data taken from the tests were examined using the chi-square and Fisher's exact and 2-tailed tests. P value less than 0.05 was determined as a significance level. Table 2 shows the population comprised in the present survey. The mean age of the studied individuals was 53.5 years. The ratio of male to female amongst the studied population was 280/430. The distribution of smoking and alcohol amongst studied patients was 44.9% and 35.2%, respectively. Among all examined clinical findings, dysuria (34.9%) was the most predominant. Table 3 represents the prevalence and antimicrobial resistance of MRSA bacteria recovered from urine specimens. Findings showed that 55 out of 710 (7.7%) urine specimens were positive for the MRSA. MRSA Table 1. Polymerase chain reaction (PCR) procedures used to detect antimicrobial resistance-encoding genes (10,11) Genes Primers ( isolates displayed the supreme resistance rate toward penicillin (100%) and ceftaroline (100%). The resistance rate against gentamicin, erythromycin, and ciprofloxacin was 87.2%, 76.3%, and 69.0%, respectively. Table 4 shows the antimicrobial resistance-encoding genes distribution amongst the MRSA isolates. BlaZ (100%) and tetK (85.4%) had the higher frequencies amongst examined antimicrobial resistance-encoding genes.

Discussion
MRSA strains are measured as one of the most critical reasons for healthcare-associated and communityassociated (CA) infections. Both CA-MRSA and HA- MRSA have the emergence of antimicrobial resistance. Reports showed that MRSA strains recovered from clinical infections displayed a considerable prevalence of resistance toward various antimicrobials classes, including cephalosporins, penicillins, quinolones, macrolides, tetracyclines, aminoglycosides, phenols, and lincosamides (12). Thus, it is essential to assess its prevalence and molecular epidemiology amongst diverse kinds of hospital infections.
The present study showed that 7.7% of the urine specimens of hospitalized patients who suffered from UTIs were positive for the MRSA strains. MRSA isolates displayed a boost resistance rate toward erythromycin, ceftaroline, penicillin, gentamicin, and ciprofloxacin antimicrobial agents. Additionally, MRSA isolates harbored a boost distribution of blaZ and tetK antimicrobial resistance-encoding genes. It seems that the antimicrobial-resistant MRSA isolates may be an emerging cause of UTIs in Iraq.
Similarly, Lunacek et al (7) labelled that the MRSA prevalence amongst urine specimens in Austria was 4.06%. They disclosed that MRSA isolates were resistant toward cephalosporin, aminopenicillin, penicillin G, carbapenem, and β-lactamase antimicrobial agents. They also presented that catheter utilization is the most critical risk factor for MRSA occurrence in UTIs. An Irish survey (13) described that the prevalence of MRSA strains was 27.9%. Besides, MRSA isolates of the urine specimens displayed the uppermost resistance rate toward flucloxacillin (100%), co-amoxiclav (100%), and ciprofloxacin (98%).
Our findings also showed the high distribution of penicillin (blaZ)-and tetracycline (tetK)-encoding genes amongst the MRSA isolates. Boost distribution of blaZ, tetK, gyrA, ermA, and msrA antimicrobial resistanceencoding genes, amongst other types of infections, has been reported from Malaysia (21), Uganda (22), and Turkey (23). There were several mechanisms of antimicrobial resistance (24). The antimicrobial resistance-encoding genes presence is one of them (25). Thus, it is not surprising that the distribution of antimicrobial resistance-encoding genes was much lower than the antimicrobial resistance pattern of the MRSA isolates toward one group of antimicrobials. However, it is essential to assess the status of antimicrobial resistanceencoding genes amongst MRSA isolates of UTIs.
The present survey was limited to the low groups of examined patients and the absence of assessing the distribution and antimicrobial resistance of MRSA amongst patients with different clinical signs of UTIs.

Conclusion
In conclusion, MRSA strains are considered an opportunist cause of UTIs in Iraq hospitals. According to findings, penicillin, ceftaroline, gentamicin, erythromycin, and ciprofloxacin prescription can not effectively be controlled and treat the MRSA's UTIs in Iraq. However, further surveys should perform to assess other epidemiological features of MRSA in UTIs.

Limitations of the study
The present study was limited to the lack of microbial assessment of urine samples of healthy volunteers as a control group, low numbers of isolated bacteria, and finally, the absence of the disk diffusion analysis of other antibiotic agents.
Authors' contribution RAK, NA, BWH and MFN were the principal investigators of the study. RAK and BWH carried out the samples collection, bacterial isolation and disk diffusion. NA carried out the MRSA identification and DNA extraction. MFN designed and supported the study and carried out the PCR genetic alignment. FE participated in statistical analysis. All authors participated in preparing the final draft of the manuscript, revised the manuscript and critically evaluated the intellectual contents. All authors  have read and approved the manuscript's content and confirmed the accuracy or integrity of any part of the work.

Conflicts of interest
The authors declare that they have no competing interests.

Ethical issues
The research followed the tenets of the Declaration of Helsinki. The present examination was performed on the urine specimens of volunteer patients hospitalized in the hospital due to UTIs. Written informed consent was taken from all participants before any intervention. Personal information of the individuals of the study is kept secret. Additionally, ethical issues (including plagiarism, data fabrication, double publication) have been completely observed by the authors.

Funding/Support
Funding information is not available.