Prevalence of Methicillin-Resistant and Methicillin Sensitive Staphylococcus aureus Nasal Carriage and their Antibiotic Resistant Patterns in Kirkuk City, Iraq

The carriage of community-acquired methicillin-resistant S. aureus (CA-MRSA) has become a source of community-associated infections, particularly in the anterior nares of people worldwide. This study was aimed at determining the prevalence of community-acquired S. aureus (CA-SA) and CA-MRSA nasal carriage among healthy individuals of various ages in Kirkuk City and evaluating the susceptibility of isolates to various antibiotics. A total of 597 healthy individuals were recruited in the community between December 1, 2021, and December 30, 2022. Nasal swabs obtained from participants were taken to the laboratory, where bacteria were isolated and identified using phenotypic characteristics. The MRSA isolates were identified by applying the modified Kirby Bauer disc diffusion technique. The results showed that the prevalence of CA-SA and CA-MRSA nasal carriers was 16.6 and 4.5%, respectively. The average age of the participants was 33.2 years, with a male-to-female ratio of 1.1: 1. The highest resistance of the isolates was observed against oxacillin (27.3%), followed by penicillin G (24.3%), amoxicillin (15.2%), erythromycin (12.1%), and tetracycline (6.1%). There was a 9.1% resistance rate to clindamycin, rifampin, gentamycin, and ciprofloxacin. However, all CA-MRSA isolates were multi-drug resistant. However, all the isolates were sensitive (100 %) to vancomycin, linezolid, and mupirocin. The findings of the present study highlight the potential for CA-SA and CA-MRSA acquisition in this population, which may be related to antibiotic abuse or overuse as well as poor hygiene. To lessen the impact of community-associated strains of MRSA nasal carriage, this necessitates the probable need for infection prevention measures and adequate antibiotic therapy.


INTRODUCTION
Staphylococcus aureus is a human skin and mucosa commensal. 1It is a major pathogen found in both the community and hospitals that cause a variety of diseases, 2 including recurrent tonsillitis, 3 pneumonia, soft tissue infections, urinary tract infections, and bloodstream infections. 2 Although S. aureus can colonize various parts of the human body, the anterior nares are the principal ecological niche for this bacterium.About 20-30% of people are permanent carriers, and about 30 % are transient carriers. 4Staphylococcus aureus has become resistant to some types of antibiotics.Methicillin-resistant S. aureus (MRSA) strains are spread worldwide and they were found to be resistant to various Beta-lactam (β-lactam) antibiotics such as penicillin, cephamycins, and cephalosporin, 5 except ceftaroline (fifth-generation cephalosporins), which is used for treating MRSA infection. 6Also, the species of Staphylococcus often developed resistance to different classes of an antibiotic such as quinolones, macrolides, and aminoglycosides. 5he mecA gene, which encodes a penicillin-binding protein with a low affinity (PBP2a), is the genetic determinant of methicillin resistance in MRSA.The staphylococcal cassette chromosome mec (SCCmec) types IV and V, which are frequently carried by CA-MRSA, are the mobile genetic elements by which the mecA gene is transported. 6Since the 1990s, there has been an increase in CA-MRSA infections in the general population, and the incidence of S. aureus and MRSA is also rising rapidly. 7,8The nasal carriage rate of S. aureus has been frequently employed as an indicator for determining the antibiotic resistance of S. aureus and MRSA in the community because it is a significant risk factor for a wide range of staphylococcal infections. 9A research in Kirkuk, Iraq, found that 16% of 100 restaurant employees had MRSA nasal carriage. 10dditional research on CA-MRSA nasal carriage has been conducted in other regions of Iraq among a variety of populations, including healthy individuals in Diyala City, 11 intermediate students in Muthanna Province, 12 healthy children in Basrah City, 13 Syrian refugees in Duhok City, 14 and secondary school students at Duhok City. 15They demonstrated relative prevalence rates of 21.5, 24, 41.2, 35, and 2.04%, respectively.Similarly, a greater prevalence of MRSA nasal carriage (55/181) among the healthy population has been recorded in other countries, such as west Iran. 16Additionally, in a community-based study conducted in Arak, central Iran, 17 Turkey, 18 Jordan, 19 and Saudi Arabia, 20 the nasal colonization rates of MRSA were reported to be 4.5, 9, 40.9, and 25%, respectively.Screening enables the identification of nasal carriers, which enhances the application of decolonization and other preventive measures to reduce postoperative infection in carriers and the use of antibiotics. 21,22herefore, the present study was conducted to determine the prevalence of CA-SA, including CA-MRSA nasal carriage, among a healthy population of all ages in Kirkuk City, Iraq.The antibiotic susceptibility of the test isolates was also detected.

MATERIALS AND METHODS
This study was conducted between 1 st December 2021 and 30 th December 2022 among healthy people living in Kirkuk City.Ethical approval was obtained from the Ethics Committee of the College of Nursing, University of Kirkuk.A total of 597 individuals of both genders, with ages ranging from 10 to 70 years, participated in the study.An anonymous questionnaire that asked about socio-demographic information like age, gender, and whether or not the respondent had ever used antibiotics or been hospitalized within the previous three months was created.The study's participants were chosen from a pool of community members.After giving their consent, approved by each participant or, in the case of minors (< 18 years old), by their parents on behalf of the minor, they were enrolled at their workplaces or homes.

Sample collection
Sterile cotton swabs were used for a biological sample collection from the anterior nares of all participants.The procedure was repeated after a month to demonstrate a persistent carrier of S. aureus, according to Azis et al. 23 The samples were rapidly transported using Cary Blair transport media to the laboratory at the Department of Medical Laboratory Techniques, College of Health and Medical Techniques, Northern Technical University, Kirkuk, Iraq.

Microbiological analysis
The samples were cultured on 5% sheep blood agar and incubated at 37°C for 24-48 hr.The isolates were sub-cultured on mannitol salt agar, and nutrient agar, and then incubated at 37°C for 24-48 hr.Identification based on characteristics of colony features, gram staining reactions, and biochemical analysis including catalase, and coagulase tests (slide and tube methods) were used to identify S. aureus isolates according to previously described procedures. 24More so, the API-Staph system (Biomerieus, France) was employed for a confirmatory test.

Antimicrobial susceptibility testing
A suspension colony of each confirmed S. aureus isolate was made in sterile normal saline and cultured at 37°C for 15 minutes.The concentration was adjusted to 0.5% McFarland Standard.Then, using the modified disc diffusion (Kirby-Bauer) technique, the sensitivity of the test isolates was assessed against a variety of antibiotics, including penicillin (P; 10 µg), amoxicillin (AM; 10µg), oxacillin (OX; 1 µg), gentamycin (Gen; 10 µg), clindamycin (CD; 2 µg), rifampin (RM; 5 µg), erythromycin (E; 15 µg), ciprofloxacin (CIP; 5 µg), tetracycline (TE; 30 µg), linezolid (LZ; 30 µg), mupirocin (200; µg), and vancomycin (VA; 10 µg) obtained from Bioanalysis Company.The results of the antibiotic susceptibility patterns were interpreted according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). 25All oxacillin-resistant isolates were assessed as MRSA by their ability to grow on Muller Hinton agar containing (6 mg/ml) of oxacillin and 4% Nacl. 26Staphylococcus aureus strain (ATCC 25923) was used as a positive control to evaluate the effectiveness of microbiological and antibiotic sensitivity testing. 27D-test was used for detecting inducible clindamycin-resistant bacteria by placing clindamycin and erythromycin discs (Bioanalysis Company) on Muller-Hinton agar plates at a distance of 15 mm from each other and were incubated at 37°C for 24 hr.Clindamycin resistance is thought to be inducible when there is a flattening or D-shaped zone between the clindamycin and erythromycin discs. 28All tests were repeated three times.

Statistical analysis
All the data were collected and tabulated.The people who had recently taken antibiotics were removed.The association between the variables was examined using the Chi-Square test, and p < 0.01 was considered significant.The prevalence of nasal carriage of S. aureus as well as MDR-SA was calculated as a percentage of all participants who were positive at the anterior nares during the specific period of the study.Also, the percentage of resistant bacteria for each antibiotic was calculated.

RESULTS
A total of 597 healthy individuals were examined for S. aureus carriage using nasal swabs.Their ages ranged from 10 to 70 years, with a mean of 33.2 years.The ratio of male to female was 1.1:1.There was no statistical association between gender and CA-SA nasal carriage.Additionally, 99/597 (16.5%) persistent S. aureus carriers were identified because they produced positive results during both the first and second sample collection events, as shown in Tables 1 and 2. MRSA and MSSA were found to be present in 27/99 (27.3%) and 72/99 (72.7%) of the participants, which constitute 4.5 and 12%, respectively.The majority of CA-SA and CA-MRSA nasal carriages, which account for 3.69 and 1.17%, respectively, of all individuals, were also found among participants aged 21 to 30 years (Table 2).This result indicates a statistical association between age and CA-MRSA nasal carriage rate.

DISCUSSION
S. aureus is one of the most common opportunistic pathogens in the community and hospitals. 2Clinically important infections are frequently caused by it, and their severity can range from mild skin infections to more dangerous invasive illnesses. 29According to earlier studies, nearly 30% of healthy individuals are permanent nasal carriers of S. aureus, with a higher rate among children. 30MRSA strains in particular pose a significant risk for the development and subsequent insidious staphylococcal infections. 31n the current study, the prevalence of persistent nasal carriage of S. aureus among the population in Kirkuk City was 99/597 (16.6%).This observation is in line with that of other investigators in Marrakesh, who found that S. aureus was present in the anterior nares of 16.3% of 300 healthy children. 32he adult population of nine European countries was studied, and the prevalence of S. aureus was found to be greater (21.6%), ranging from 12.1% in Hungary to 29.4% in Sweden. 33Another study from the Iraqi province of Diyala also found a higher carriage rate than what was obtained in the present study, which was 21.5% out of 1186 healthy people. 11Moreover, the value obtained in the current study was found to be lower compared with previous studies in other areas of the world, such as Turkey, 18 northwest Ethiopia, 28 Iran, 16 Italy, 34 Nepal, 35 which documented a carriage rate of 17, 23, 30.6, 42, 44, and 66%, respectively.In addition, the nasal carriage prevalence of S. aureus among specific groups, including healthcare workers in Erbil City, 36 healthy children in Basrah City, 13 secondary school students in Duhok City, 15 workers in restaurants in Kirkuk City, 10 were 24.5, 14.24, 18.4, and 30%, respectively.The variation of nasal carriage of S. aureus in the different investigations could be attributed to differences in geographical distribution, properties of the studied participants, sampling methodologies, preservation, and diagnostic methods. 28It has been reported that the mucin layer in the anterior nares is related to S. aureus colonization in this area of the body through an interaction of mucin carbohydrates with staphylococcal protein. 37RSA nasal carriers were present in 4.5% of the study's participants.This result is consistent with a community-based study from Arak, Iran. 17It was greater than the prevalence rate of CA-MRSA nasal carriage among secondary school children in Duhok City, Iraq, which was reported by previous researchers to be 2.04%. 15n other studies undertaken in Iraq, the nasal carriage prevalence of CA-MRSA among healthy children in Basrah City, 13 intermediate students at Muthanna Province, 12 and healthy workers in restaurants at Kirkuk City, 10 were 41.2, 24, and 16%, respectively.Furthermore, the result of the current study is consistent with the reports from Italy, 34 India, 38 and Marrakesh, 32 which showed that the prevalence of MRSA nasal carriage was 3, 3.17, and 4%, respectively.As opposed to reports from Egypt (32%), Saudi Arabia (25%), 20 Africa (15%), 28 Northwest Ethiopia (9.79%), 28 and Jordan (40.9%), 19 the prevalence of MRSA carriage in the present study was lower.The differences in the nasal carriage rate among these populations might be attributed to the initiation of antimicrobial therapy before sample collection. 39Furthermore, some research has discovered a link between S. aureus resistance to methicillin and the use of a beta-lactam combination. 40oncerning gender, the result of the current study showed that the percentage of male carriers was higher than that of females, but there was no statistical association between gender and MRSA carriage rate (p<0.01).This result is consistent with that reported by Abdelmalek et al. 19 However, there is a significant statistical association between age and the CA-MRSA nasal carriage rate, as most CA-SA, and CA-MRSA nasal carriers were among the ages of 21 to 30 years, which constitute 3.69, and 1.17%, respectively, of all the participants.This observation might be attributed to attendance at a fitness center, as this practice is becoming very common in Iraq, particularly among males.This result is consistent with a recent study conducted in Jordan that examined the link between S. aureus carriage and gym attendance.Regarding antibiotic susceptibility, the majority of the studied isolates were resistant to oxacillin (27.2%), followed by penicillin G, 24/99 (24.3%), and amoxicillin, 15/99 (15.2%).The values obtained in this result were lower than those of other studies involving healthy individuals from the community, which showed high resistance to penicillin of about 65%, 23 resistance to penicillin in Europe (87.1%), 41and resistance to penicillin in Nepal and Ohio (100%), with only 5.8 and 1.1% resistant rates to oxacillin in Nepal and Ohio, respectively. 35The resistance of S. aureus isolates to penicillin was determined within a decade after its introduction in 1940. 42As a result, new betalactam antibiotics were produced, which were related to the appearance of S. aureus strains that produce beta-lactamase. 43Since methicillin was developed treat infectious disorders brought on by microbial strains resistant to penicillin, the occurrence of resistant S. aureus to methicillin has resulted in a decrease in the effectiveness of antibiotic therapy. 44Lower resistance of isolates was shown to exist against erythromycin (18.1%) and tetracycline (12.1%).Other studies also reported a low resistance (<12%) to both tetracycline and erythromycin, which is in line with the results obtained in the present study. 23Other investigators showed in a comparative study of two cities in Egypt and Saudi Arabia that a higher resistance rate to erythromycin was observed in selected MRSA and MSSA isolated from outpatients.Values of 55 and 24% were recorded for MRSA and MSSA, respectively, in Egypt and 58 and 25%, respectively, in Saudi Arabia. 20the percentage of resistance to clindamycin, rifampin, gentamycin, and ciprofloxacin was 9.1%.This observation was in line with other studies that reported low resistance of their S. aureus isolates and recorded high sensitivity of 94, 98, 90.2, 84 and 93% to clindamycin, gentamycin, cefoxitin, cotrimoxazole, and ciprofloxacin, respectively. 28omparatively, all studied isolates (100%) were sensitive to vancomycin, linezolid, and mupirocin, indicating that these are appropriate treatments for MRSA infection and decontamination of MRSA carriers.This result is consistent with other findings in Basra, Iraq, that reported 100% susceptibility to vancomycin. 45n addition, other researchers from Erbil, Iraq, demonstrated that all isolates of MRSA and MASSA were susceptible to linezolid and mupirocin. 36The results also revealed that MRSA accounted for 27.3% of the isolates of S. aureus from healthy people's anterior nares, which were 33.3% multi-drug resistant.This observation is slightly higher than another study, which reported 30.8%. 28Onanuga and Temedie, on the other hand, found that 52.5% of their isolates were multi-drug resistant community-associated S. aureus infections, 46 of which were higher than the result of the present study.Moreover, 2.51% of isolates showed an inducible clindamycinresistant phenomenon.This observation was lower than that reported by Baguma, who discovered that 42% of the test isolates showed this phenomenon. 47There are many recommendations for applying the PCR techniques in the diagnosis of Methicillin-resistant Staphylococcus aureus 48,49 due to their accurate results and short time.[63][64]

CONCLUSION
The findings from this study reveal that there is a chance that the population under study will develop community-associated S. aureus, MRSA, and multidrug resistance isolates, which may be caused by improper or excessive use of antibiotic therapy and poor hygiene.Additionally, it has been observed that MRSA isolates had a greater rate of antibiotic resistance than MSSA isolates during the investigation.This calls for prospective surveillance among local populations in various parts of the country, as well as molecular characterization of nasal carriage CA-MRSA to ascertain the prevalence and profile of antibiotic resistance among local communities across the country.Also, applying antimicrobial stewardship will help reduce the burden of antibiotic-resistant bacteria and community-associated MRSA nasal carriage infections.

Table 2 .
Distribution of S. aureus and CA-MRSA nasal carriage based on age group and sex M: Male; F: Female; No.: Number: %: Percentage