Emerging Challenges in Staphylococcus aureus Bloodstream Infections: Insights from Coagulase Typing, Toxin Genes, and Antibiotic Resistance Patterns

Background The incidence of complications and mortality associated with Staphylococcus aureus (S. aureus) bloodstream infections has been increasing significantly, particularly in developing countries where control strategies against this virulent pathogen and its resistance to antibacterial agents are insufficient. The aim of this study was to investigate coagulase typing, the prevalence of toxin genes, and the antibiotic resistance profile of S. aureus isolated from bloodstream infections. Methods Antibiotic susceptibility of the isolates was determined by the disk diffusion method. The prevalence of toxin genes was determined using the polymerase chain reaction (PCR) method. Genetic variability of isolates was determined using multiplex PCR based on coagulase gene polymorphism. Results Out of 120 strains, 55 (46%) were methicillin-resistant S. aureus (MRSA) and 65 (54%) were methicillin-sensitive S. aureus (MSSA). All isolates were susceptible to linezolid and teicoplanin but showed varying levels of resistance to other antibiotics. The highest resistance was observed for ampicillin (92.5%), gentamicin (69.2%), and amikacin (68.3%). Multidrug resistance was observed in all isolates. PCR analysis revealed a higher prevalence of toxin genes in MRSA (tst: 38%, pvl: 29.1%, eta: 10%, and etb: 4.1%) than that in MSSA. According to the coa typing, the most prevalent types were coa III (29.2%), coa II (26.7%), and coa VI (10%). Conclusion The presence of genetic variability and widespread multidrug resistance in our hospitals emphasizes the circulation of various coa types. Therefore, it is crucial to implement antimicrobial stewardship and infection control measures to prevent and control the spread of these strains.


S. aureus isolated from
BSIs is now considered a highpriority pathogen with the potential to increase fatality rates globally [4][5][6].Te rising drug resistance in virulent strains, particularly methicillin-resistant S. aureus (MRSA), poses a serious challenge in the treatment and control of staphylococcal infections [7,8].Te indiscriminate use of antibiotics in both the community and hospital settings has led to increased selective pressure on S. aureus, resulting in the proliferation of antibiotic resistance [7][8][9].Evidences indicated that numerous S. aureus virulence factors including toxic shock syndrome toxin-1 (TST-1), pantonvalentine leukocidin (PVL), staphylococcal enterotoxins (SEs), and exfoliative toxins (ETa and ETb) have been associated with the pathogenesis of this bacterium [10][11][12].Several molecular typing methods have been employed to genotype S. aureus strains, including pulsed-feld gel electrophoresis (PFGE), staphylococcal cassette chromosome mec (SCCmec) typing, agr typing, protein A gene (spa) typing, multilocus sequence typing (MLST), and coagulase gene (coa) typing [7,13,14].Coa typing, a multiplex PCR-based method, is cost-efective, rapid, easily interpretable, and suitable for identifying genetic relationships among S. aureus isolates [15,16].Coagulase, an extracellular protein, plays a vital role in pathogenesis of S. aureus [16][17][18][19].Generally, the activity of the coagulase enzyme in S. aureus pathogenesis is multifaceted.It aids in immune evasion, facilitates the establishment of infection, contributes to the formation of abscesses, and promotes bioflm formation.Understanding the role of coagulase is crucial for developing strategies to combat S. aureus infections.
Ten diferent coa types (ScI-X) have been identifed based on variations in the signal sequence, N-terminal D1 and D2 regions, central region, 27-amino acid repeat regions, and C-terminal sequence [16,20].Recent studies have reported a correlation between specifc genotypes of S. aureus, multidrug-resistant (MDR) patterns, and virulence profles [2,3,6,8,9].Tere are only a small number of studies available worldwide addressing the genotyping of S. aureus isolated from blood infections [4,7].Te present study was attempted to determine the antibiotic resistance pattern, toxin profle, and molecular characteristics of S. aureus isolates obtained from patients with bacteremia.

Study Design, Sample Collection, and Ethical
Considerations.In this cross-sectional study, from August 2021 to July 2022, a total of 120 S. aureus isolates obtained from BSIs were investigated.Te study protocol received approval from the Ethics Committee of the Shahid Beheshti University of Medical Sciences in Tehran, Iran (IR.SBMU.MSP.REC.1401.714).All isolates were cultured on blood agar and mannitol salt agar and characterized by Gram staining and conventional biochemical tests, including catalase production, and coagulase and DNase tests (HiMedia, Mumbai, India).All phenotypically confrmed S. aureus isolates underwent polymerase chain reaction (PCR) assay for the nuc gene detection and fnal confrmation.All confrmed isolates were stored at −70 °C in Tryptic Soy Broth (TSB, HiMedia, Mumbai, India) supplemented with 20% glycerol for further analysis.To screen for methicillin-resistant S. aureus (MRSA) strains, a disk difusion method using cefoxitin (30 μg) on Mueller-Hinton agar was performed.Te mecA genes were detected by PCR as described elsewhere [21].

Evaluation of Toxin Gene Expression. Te PCR technique
with specifc dinucleotide primers was employed to detect toxin-encoding genes, including panton-valentine leukocidin (pvl), exfoliative toxins (eta, etb), and toxic shock syndrome toxin (tst) (TIB Molbiol, Berlin, Germany).In each run test, positive controls for toxin genes were used.Table 1 provides a description of the primers and amplifcation conditions utilized.

Coagulase Typing.
Te coa types (I-X) were analyzed using a multiplex PCR-based method with four sets (A-D) of primers and PCR conditions explained by Hirose et al. [16].Set A primers were designed to identify coa types I, II, III, IVa, IVb, and Vb.Set B primers were utilized to distinguish coa types VII, VIII, and X. Set C primers were used for the identifcation of coa types IX and Vb.Finally, Set D primers diferentiated between coa types IVa and IVb.As positive controls, standard S. aureus strains representing coa types I-X were used in each PCR assay.

Statistical Analysis.
Te data were analyzed with Statistical Package for Social Sciences (SPSS) software V22.0 for Windows.Qualitative data were expressed using numbers and percentages.

Data and Antibiotic Susceptibility.
In this study, a total of 120 S. aureus clinical isolates were collected from blood samples.Te blood cultures were incubated for 18-24 hours, and the positive cultures were cultured on blood agar and mannitol salt agar.It is important to note that only one sample was obtained from each patient, ensuring that the isolates were nonduplicate.One sample was obtained from each patient.Te exclusion criteria for this study involved the presence of bacterial species other than S. aureus in the blood cultures, as well as contaminated blood cultures.Te mean age of patients was 46.6 years, ranging from 1 to 69 years old, with 47.5% (n � 57) being male and 52.5% 2 Advances in Medicine Te antimicrobial susceptibility testing revealed the highest resistance rate for ampicillin (92.5%), followed by gentamicin (69.2%), amikacin (68.3%), erythromycin (61.7%), tetracycline (46.7%), kanamycin (39.2%), clindamycin (45.8%), chloramphenicol (20%), cephazolin (20%), ciprofoxacin (42.2%), rifampin (32.5%), and trimethoprimsulfamethoxazole (13.3%) (Table 2).All of the isolates were found to be MDR based on the antimicrobial susceptibility testing.As given in Table 3, the most prevalent resistance profle among the MDR isolates was resistance to 3 antibiotics (24.2%), followed by resistance to 4 antibiotics (20.8%), 6 antibiotics (20%), and 10 antibiotics (11.4%) simultaneously.In our study, eleven resistance patterns were detected, wherein ERY, AMP, and AMK (8.3%) and AMP, CIP, CEF, GEN, ERY, and CLI (15%) were the top frequently identifed profles in MSSA and MRSA strains, respectively.

Distribution of Virulence Encoding Gene.
In this study, the tst gene was confrmed in 38.3% of the isolates but the pvl gene was present in 29.2%, eta in 10%, and etb in 4.2% of tested isolates.Te prevalence rates of toxin genes were higher in MRSA strains compared to MSSA strains.S. aureus isolates carrying pvl genes had resistance to 6 and 4 antibiotics simultaneously, while TST-positive isolates indicated resistance to 3, 4, and 6 antimicrobial agents.

Discussion
Microbial resistance of S. aureus, especially MRSA, is a serious problem for human health and patients worldwide.As MRSA causes bloodstream infections in hospitalized patients, the increased antibiotic resistance in this bacterium is a signifcant threat [22,23].Multidrug resistance has been recognized as a major issue in healthcare management due to the consequences of inadequate infection control measures [24].Several studies have investigated the resistance rates of S. aureus isolates from blood samples [7][8][9], and the present study reports similar fndings among patients with bacteremia at diferent wards of Shahid Beheshti University of Medical Sciences hospitals.Te frst aspect we assessed was the sensitivity of the isolates to linezolid, an advanced antimicrobial agent known for its activity against S. aureus isolates.In line with fndings from previous research [24][25][26], the present study showed that all the isolates were sensitive to linezolid.Tis fnding suggests that linezolid may be a potential treatment option for patients infected with MDR S. aureus strains [27].
Tere is considerable data on the prevalence rate of MRSA strains isolated from BSIs across the world.In line with our results, a high prevalence of MRSA strains was also reported in earlier research conducted in diferent geographical regions [22][23][24][25].Our study revealed a high prevalence of resistance to many antibiotics commonly used for bloodstream infections (BSIs).Interestingly, no resistance to teicoplanin and linezolid was observed, consistent with fndings reported by Alharbi [25].Conversely, the highest resistance was detected against ampicillin and amikacin.Furthermore, all analyzed isolates were classifed as MDR, indicating a particular risk to public health.Tese fndings emphasize the urgent need for implementing appropriate antibiotic stewardship programs and the use of specifc treatment protocols in patients infected with these strains to mitigate the spread of MDR S. aureus.
Te study also reported a high percentage of MDR among MRSA strains compared to MSSA.Various studies from diferent regions have also reported similar fndings [28,29].A similar survey conducted by Eslami et al. on 59 S. aureus isolated from wound showed that the prevalence of MRSA (59.3%) was greater than that of MSSA (40.7%) [13].Soltani et al. recently performed an epidemiological analysis on 95 S. aureus and investigated the MDR in MRSA and MSSA strains isolated from diferent clinical samples.Tey reported a higher MDR rate in MRSA strains compared to MSSA (87.5% vs 77.4%) [3].A study by Rahimi et al. focused on 419 clinical S. aureus strains isolated from urinary tract infection.Tey indicated that 25.8% and 74.2% of tested isolates were MRSA and MSSA, respectively.Also, all the strains were to be susceptible to linezolid, vancomycin, penicillin, and tigecycline [11].Recent evidence has highlighted the pivotal role of toxins as a major virulence factor in the pathogenesis of S. aureus-induced infections [26].According to our data, 38.3%, 29.2%, 10%, and 4.1% of S. aureus strains were found to carry tst, pvl, eta, and etb encoding genes, respectively.Tis result is similar to that of the Iranian study by Goudarzi et al., who indicated that the tst gene was the predominant type in S. aureus strains (58.6%), followed by pvl (19.5%), eta (7.8%), and etb (5.5%), respectively [8].A study conducted by NI Ahmad et al. [28] in Malaysia reported that only 20% of MRSA strains were positive for the pvl gene, which was in contrast with our results.However, similar to our fndings, the prevalence rate of the pvl gene in MRSA strains was higher than that in MSSA strains.Diferences in the prevalence of the pvl gene among S. aureus strains may be infuenced by factors such as 4 Advances in Medicine origin of isolates, clinical samples, and geographical factors and, most importantly, the presence of diverse PVL-carrying phages within S. aureus strains.
Our results, in relation to tst gene, coincide with those produced by Soltani et al. in Iran who reported tst gene in 18.9% of S. aureus strains isolated from clinical samples [3].Similarly, in a systematic review and meta-analysis study conducted in Iran, Shahini Shams-Abadi et al. reported a relatively high carriage rate of tst gene (21.3%)[29].In a similar survey conducted in Korea by Kim et al., on 576 S. aureus strains isolated from BSIs, showed that 26.8% of the examined isolates were tst positive [30].Possible explanations for this raised prevalence are the genetic diversity of bacteria, high expression levels of this gene in BSIs, and the transmission of these genes among diferent isolates.
In our study, the prevalence of the eta and etb genes was found to be relatively low, consistent with the fndings reported by Tayebi et al. [31].Tis indicates that the presence of these exfoliative toxin genes may not be signifcantly associated with bloodstream infections.Furthermore, the low prevalence of these exfoliative toxin genes may suggest that other more virulent factors are contributing to bloodstream infections in this population.

Advances in Medicine
As presented in Table 4, 10 diferent coa types are detected in this research.In agreement with the published data by Hirose et al. [17], which indicated genetic variability of S. aureus in Japan, the current research indicated genetic diversity of S. aureus isolated from blood infection with the predominant genotype of coa III (29.2%).Likewise, a study by Afrough et al., conducted in Iran, examined 157 clinical isolates of S. aureus and revealed a high prevalence rate of MRSA (83.7%), as well as genetic diversity in coa genes.Te study identifed six diferent coa patterns, with the C1 pattern being the most common (21.7%) [18].Another study conducted by Goudarzi et al. in Iran, on 89 MRSA strains from burn patients, indicated 10 coa types with the majority of type III (47.2%) [32].A similar survey conducted by Mohajer et al. in Iran on 258 S. aureus isolates indicated fve coa types [33].In an Egyptian study conducted by Abdulghani and Khairy on 58 MRSA strains recovered from clinical isolates, 15 diferent coa types with the prominence of coa type X (16.6%) were identifed [34].In a Sudanese study conducted by Ibrahim et al., resistance to methicillin was found to be 56% and coa type III as the predominant type was reported (55.5%) [35].

Conclusion
Tis study provides valuable insights into the prevalence of antibiotic resistance, toxin gene expression, and coagulase types among S. aureus strains causing bacteremia.Te genetic diversity of the coa gene, with the prominence of coa type III and high MDR, poses potential risks in our healthcare settings, emphasizing the need for proper and comprehensive approaches for systematic surveillance to reduce the rate of infections.Continuous monitoring of the antimicrobial susceptibility surveillance system and antibiotic stewardship programs (ASPs) are necessary for better patient management.Te observed genetic variability highlights the signifcant health risk posed by S. aureus in patients with bacteremia.

Table 1 :
Te PCR conditions and oligonucleotide primers used in this study.� 63) being female.Te occurrence of S. aureus bacteremia was more frequent in the age group 30-70, with the lowest occurrence observed in age groups under 10 and above 60.Te distribution of S. aureus isolates in various hospital wards was as follows: intensive care unit (ICU)

Table 2 :
Antibiotic resistance pattern of 120 S. aureus isolates collected from blood samples.

Table 3 :
Distribution of MDR patterns in MRSA and MSSA strains isolated from blood samples.

Table 4 :
Distribution of diferent coa types in S. aureus strains obtained from 120 patients with bacteremia.