The First Report of t8463 and t605 spa Types in Methicillin-resistant Staphylococcus aureus Isolated from ICUs in Rasht, Iran

1. Introduction

In the last several decades, Staphylococcus aureus has been one of the most important human pathogens and the leading cause of acquired infections in the community and hospital. S. aureus causes a wide range of diseases in humans, from mild skin infections to life-threatening diseases. This pathogen exists in the nasal and intestinal cavities of people and is colonized in approximately 50% of healthy people in the community and hospital personnel without symptoms ( 1 ). Although there are bacteria in the body of healthy people, antibiotic resistance can lead to severe infections and even death. S. aureus becomes resistant to most antibiotics in a short period and causes many treatment problems. There are many virulence factors associated with S. aureus, including pathogenicity and bacterial colonization ( 2 ). Methicillin-resistant S. aureus (MRSA) is the cause of various types of antibiotic-resistant infections and is one of the most important and common resistance patterns among S. aureus strains. This resistance is caused by the presence of the mecA gene, which is chromosomally encoded ( 2 , 3 ). The mecA gene and its expression-regulating genes and additional sequences form a gene complex called the mecA complex on a very large gene island called Staphylococcal Cassette Chromosome mecA (SCCmecA), which can transfer between different strains and leads to the spread of resistance ( 4 ). The prevalence of MRSA varies widely in different geographical regions and has shown a significant increase worldwide in recent years. The prevalence of MRSA strains in Asian countries such as China, Korea, and Taiwan is more than 70%; in North America, it is more than 50%; in Europe, it is 20%; and in Iran, it is on average 50% ( 5 ). Different regions of Iran have also been reported in various ranges between 20 and 90 percent for the prevalence of MRSA ( 6 ). The prevalence of MRSA is higher, especially in nosocomial infections, patient care centers, patients with open wounds, patients with prostheses, and also in immunodeficient patients. Despite the antibiotic resistance pattern in MRSA isolates and the widespread appearance of multi-drug resistant (MDR) MRSA, it is important to periodically evaluate the antibiotic resistance pattern using sensitivity tests ( 7 ). Genotyping can be extremely useful for identifying the source of infection and controlling it. Also, typing of S. aureus isolates is a suitable method for epidemiological purposes. One of the methods to describe the specific genetic traits of MRSA is to determine the type of SCCmec based on the type of recombinase enzyme encoded by ccr genes and mecA complexes. So far, 11 different types of SCCmec have been known, of which five types SCCmec I-V are dominant in Iran ( 8 ). Another method is spa typing. The spa gene encoding protein A (Staphylococcal protein A; spa) is one of the determinants of differentiation, which has the x polymorphism in a short sequence region, and different studies have identified various patterns of this gene. It can also be used to determine the specific identity of this bacterium ( 9 ). As the most important human pathogen, the cause of community- and hospital-acquired infections, the high colonization rate in medical personnel and the importance of carriers in the transmission and circulation of infection in the hospital, in this study, the origin of MRSA colonies was determined by the spa method from the personnel, patients, surfaces, air, and devices in the ICU, of Velayat and Poursina Hospitals of Rasht.

2. Materials and Methods

2.1. Sample collection

In this cross-sectional study, 500 swab samples were collected from patients, personnel, beds, clothes, and other objects in the ICUs of Rasht Velayat and Poursina Hospitals over eight months, once every two weeks. To take samples from the patients, first, using the information from the patient's file, a checklist containing age, sex, history of antibiotic use, clinical symptoms, and samples were taken depending on the type of infection, from pus, catheters, wounds, urine, blood, eye secretions also hands (palm and dorsum, under the nail, and between the fingers), etc. Also, the air of the ICU ward was sampled using the Andersen pump and S. aureus species were investigated using standard microbiological methods.

2.2. Antibiotics susceptibility test

The antibiotic resistance pattern of the isolated strains was investigated using antibiotic discs of erythromycin, clindamycin, gentamicin, linezolid, tetracycline, rifampin, penicillin, and mupirocin according to the Clinical and Laboratory Standards Institute (CLSI). The 30 µg cefoxitin disk was used to evaluate methicillin resistance and detect MRSA strains. The E-test strip was also used for vancomycin. The genome of S. aureus strains was extracted from 24-hour bacterial cultures according to the instructions of the manufacturer of the Roche kit in Germany. The oligonucleotide primers were used to carry out the standard PCR reaction to amplify the spa gene (Table 1).

2.3. Detection of resistant genes

Polymerase chain reaction (PCR) reaction was performed using 2x Super PCR Master mix (Dye Plus), 10 pmol from each primer, ddH2O, and 50-100 µmol DNA. The PCR product was electrophoresed on 1.5% agarose gel ( 10 ). The different stages involved in PCR reactions include pre-denaturation, denaturation, annealing, extension, and final extension (Table 2). The S. aureus LMG 8224 strain was used for the nucA gene and the S. aureus ATCC 33591 strain was used for the femA and mecA genes as positive controls.

2.4. The spa typing

PCR amplification was performed for spa typing of isolates (Table 2). The spa typing of MRSA strains was performed after determining the sequence of the amplified protein A gene through PCR (provided by Fazapzoh Company), utilizing specific primers available at https://spaserver.ridom.de/). The analysis was conducted using Chromas and Nucleic Acid Sequence Massager software.

2.5. Statistical analysis

Data were analyzed by SPSS version 26 software. The relationship between the variables was checked using the chi-square test and the significance level was considered to be <0.05.

3. Results

In the present study, from 500 swabs collected from patients, personnel, and fixed surfaces of the ICUs in the hospitals of Velayat and Poursina in Rasht, 45 (9%) isolates were identified as S. aureus. Among all the studied samples, 14 (31.11%) and 26 (57.78%) isolates belonged to females and males respectively. Also, 5 (11.11%) isolates were isolated from surfaces, but none from the air. Among the male participants, 25 (55.5%) were isolated from male patients and 1 (2.2%) from personnel, and among female participants, 8 (17.7%) were isolated from patients as well as 6 (13.3%) were isolated from personnel. Most samples were obtained from the hands, noses, wounds, beds, and clothes of patients and personnel, as well as from other devices in the ward. Out of 45 S. aureus isolates, 8 (17.8%) were isolated from the burn ICU at Velayat, 22 isolates (48.9%) from the general ICU, and 15 (33.3%) from the trauma ICU at Poursina Hospital. The most strains were isolated from the hand and nose of the trauma ICU, while the lowest was isolated from the burn ICU. In addition, the strains isolated from the wound were the most frequent in the general ICU, and no strain was isolated from the burn ICU of Velayat Hospital. The most frequently isolated strains came from beds and clothes of patients, uniforms of personnel in general, trauma and burn ICUs at 42.9%, 33.3%, and 23.8%, respectively. There were no isolates of S. aureus strains in trauma ICU devices and air, but most samples (three samples) were from general ICU devices. The PCR reaction for the nucA gene was carried out using specific primers. Although 65 (13%) of the samples showed infection with S. aureus in the phenotypic test, 45 isolates were confirmed with the nucA gene. penicillin, erythromycin, and penicillin antibiotics showed the highest resistance in the General ICU, Trauma ICU, and Burn ICU, respectively. However, linezolid had the lowest resistance in all three sections, and all isolates (100%) were resistant to this antibiotic. Among the isolated strains, 31 (68.9%) isolates were resistant to cefoxitin that all of them were positive for the mecA gene in the diagnosis. The MIC results of vancomycin resistance in S. aureus isolates indicated that all isolates were sensitive to vancomycin. The strains were divided into two groups: those resistant to methicillin and those susceptible to methicillin. Resistance to all antibiotics in the trauma ICU ward except penicillin and mupirocin was higher than other ICU wards. This study displayed different patterns of antibiotic resistance to various antibiotics (Table 3). The PCR reaction for the femA gene, which is a regulatory gene for methicillin-resistant strains, was performed using specific primers. All studied strains, including methicillin-resistant and methicillin-susceptible strains, were examined for the presence of the mentioned gene. This gene was detected in 31 (68.8%) isolates. There was no statistically significant difference between the presence or absence of the femA gene in MRSA and MSSA groups using the Chi-square test (P value = 0.540). Finally, all the isolates with the mecA gene were the same samples that were phenotypically resistant to the methicillin. Among the examined samples, three different spa types were detected in this work that spa type t14870 was dominant (95.5%). Also, two types, t8463 (2.2%) and t605 (2.2%), were detected for the first time in Iran and were observed only in two strains, both of which were MRSA and were obtained from the patients' noses (Table 4).

4. Discussion

S. aureus is a bacterium that colonizes on the skin and nasal mucosa and has genes that play a role in increasing antibiotic resistance prevalence. As carrier personnel can transmit the organism and cause severe nosocomial infections in hospitalized patients, it is necessary to prevent and control this infection. Recently, there have been many indications that the environment can play an important role in the spread of S. aureus. Due to its ability to adapt to various environmental conditions, including humidity, temperature, levels of ultraviolet radiation, and the type of disinfectant used, the bacterium remains stable in dry environments ( 2 ). With the increase of the elderly in society, more people are exposed to surgeries and invasive methods with medical devices, resulting in the spread of diseases in immunodeficient patients; therefore, eliminating S. aureus from the hospital environment is important to preventing and reducing nosocomial infections ( 2 ). On the other hand, determining the antibiotic resistance pattern of the isolated S. aureus and selecting the appropriate drug will reduce treatment costs and promote the rapid recovery of the patient ( 2 ). In the present study, 14 (31.11%) S. aureus samples were isolated from females, 26 (57.78%) samples collected from males, five (11.11%) isolates were isolated from surfaces, and no isolate was isolated from the air. Among the male participants in the study, 25 were isolated from male patients, and one was isolated from personnel. Among the female participants, eight isolates were obtained from patients and six isolates were obtained from personnel. In this regard, studies have been conducted that show the frequency of S. aureus isolated from medical workers was 25.2% in Hamadan ( 11 ), 31% in Tehran ( 12 ), 42% in Babol ( 13 ), and 36% in Ghaemshahr ( 14 ). The difference in the number of carriers in different Iranian studies may be attributed to environmental factors such as the long presence of medical staff in hospital wards and the direct contact of medical staff with patients, whose exposure is greater than that of students. Our data showed that the frequency of S. aureus in the nose of the carriers was observed in 26 (57.7%) isolates, which was highly similar to the study by Sharma et al ( 15 ). Also, there have been different reports of the prevalence of S. aureus in health workers in other parts of the world that rate of S. aureus in the nose of the carriers was 74.6% which was higher than our study ( 16 ). Although the prevalence of S. aureus was reported as 18.3% in the nose of healthcare workers ( 17 ), another study reported 52.2% of healthcare workers were S. aureus carrier ( 18 ). It seems that this difference in the frequency of S. aureus carriers in the nose of hospital personnel can be related to the differences in infection control committee programs and monitoring systems to identify and treat carriers, as well as the differences in race and geographical area. Our results showed that among S. aureus isolates, 68.9% were MRSA. The prevalence of MRSA carriers has been different in areas of Iran, Pourramezan et al. reported 22.5% of MRSA among health-care workers of critical wards at a university hospital of Tehran ( 19 ) but Abdullahi et al. found MRSA in 75% of healthcare workers in southwestern Iran ( 20 ). Similar to the results obtained from our study, Ghasemian et al. identified a high rate of MRSA (74.2%) in patients on hemodialysis ( 14 ). Contrary to our results, in a study conducted in Birjand, the prevalence rate of MRSA in the hospital was declared as 39.4%, which is much lower than the prevalence rate obtained from our study ( 21 ). These differences can be attributed to the study population, the type of clinical isolates, and the prescription of specific antibiotics in different geographical areas. In other parts of the world, there are reports of different levels of MRSA, in this regard, a study conducted in India on medical students to isolate MRSA revealed that only one of one hundred eighty one medical student was carrying MRSA ( 15 ). In accordance with our results, a study conducted in Brazil reported a high frequency of MRSA (72.3%) in the nose of carriers in primary health care units ( 16 ), but in Nepal 47.4% MRSA detected in nasal carriage among health care workers and medical students ( 17 ). A study in Nepal showed that 36 (92.3%) of 39 S. aureus isolated from healthcare staff in the ICUs were declared as MRSA ( 18 ). This difference seems to be related to the sample size, the geographical distance between the sampling locations, the type of sample and dissemination of specific clones. The predominant spa type in our study was t14870 (95.5%) and two spa types including t8463 (2.2%) and t605 (2.2%) were found for the first time in Iran, both of which were MRSA as well as were obtained from the noses of patients. In this regard, Gudarzi et al. performed spa typing among 90 samples S. aureus which identified five spa types including t037, t030, t790, t969, and t044 ( 22 ). Similar to the results obtained with our study, some studies in Iran have shown that t14870 is the most common spa type among S. aureus, although Montazeri et al. in Ahvaz ( 23 ) introduced different spa types, were mentioned t14870 was predominant spa type. Contrary to our results, other studies have introduced other spa types as the most common spa types in Iran, and in Hashemizadeh et al. different were identified spa types (spa003, t386, t1877, t314, t030, t186, t1816, t304, t325, t345), of which spa type t030 was the most common among only MRSA isolates ( 24 ). In another study, Latifpour et al identified 27 different spa types in only hospital MRSA isolates, and type t030 was introduced as the most frequent spa type ( 25 ). To the best of our knowledge, these spa types may be different according to different geographical regions in different countries or even different cities, different type of sample (For example, some studies have only investigated spa types in MRSA, others in MSSA, and some investigated in clinical samples, but some in healthcare workers). Our results showed a high prevalence of MRSA in the carriers of healthcare workers and three spa types were identified in them. Considering the importance of MRSA in hospitals, there is a need for a new control policy to address infections caused by this bacterium in hospitalized patients and hospital personnel. Hence, identification of new spa types emphasis on the reality which periodically performed MRSA surveillance studies in order to identify major molecular spa types related to human infection and characterize the new emergence of spa types.

Acknowledgment

The authors are grateful to each person who contributed to the completion of this study.

Authors' Contribution

Writing and Investigation: P.K

Methodology, Revision and Supervision: A.M

Revision and Visualization: M.A

Writing and Interpretation of data: G.M

Statistical analysis and Methodology: M.G

Ethics

It is stated that all ethical considerations were taken into account in the preparation of the submitted manuscript.

Conflict of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data Availability

The data that support the findings of this study are available on request from the corresponding author.

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