With few available treatments, methicillin-resistant and macrolide-resistant S. aureus (McRSA) pose serious risks to human health and are characterized by limited therapeutic options [14, 31]. This study aimed to identify the respective pathogens’prevalence and susceptibility profile to various antibiotics being employed in their treatment in one of the Major tertiary care Hospitals in Egypt. In addition, we aimed to evaluate the antimicrobial activity of PST alone and in combination with other commonly used antimicrobial agents in treating McRSA and MRSA in Egypt. In the literature, limited studies are available that are concerned with examining the antimicrobial activity of PST either alone or as combination with other antimicrobial agents. In this study, a total of 154 non-clonal S. aureus clinical isolates were collected during the period from March 2022 to February 2023 and were evaluated against 22 antimicrobial agents according to CLSI guidelines [24]. Results showed that about 43% of the collected isolates were MDR and the highest non-acceptability was recorded against beta-lactams (70–87%), including cefoxitin (43.5%), penicillin (87%) as well as macrolides including erythromycin (67.5%), azithromycin (32.46%), clindamycin (35.06%), and clarithromycin (54.54%). However, the lowest resistance was noted against linezolid (5.2%), which was followed by doxycycline (14.9%), vancomycin (9.1%), teicoplanin (9.1%), and chloramphenicol (12.3%). Our findings are in alignment with the findings of other recent studies [32,33,34,35]. Based on these findings, further investigations are urgently required to find the solution to combat the rise in antimicrobial non-susceptibility, particularly toward beta-lactams and macrolides, being developed by S. aureus clinical isolates. A few years after macrolides were introduced into the treatment, the first staphylococcal strains resistant to these antibiotics emerged. Currently, MAC resistance is common globally, and many bacteria are resistant to MACs, lincosamides, and streptogramin type B (MLSB) antibiotics [36]. The rising MAC resistance among staphylococci could be attributed to their frequent use in our clinical setting, which is usually coupled with the resistance to lincosamides and streptogramins as previously reported [31].
As previously reported, there are three primary mechanisms of MLSB resistance in staphylococci, including target site modification, efflux of MAC outside the cell, and enzymatic inactivation. Nevertheless, only the first two play a significant role in S. aureus resistance [37]. Because the MDR S. aureus isolates exhibited 100% resistance to erythromycin, clarithromycin, and cefoxitin, they were selected for testing three of the most common MAC-resistant genes. Four clinically relevant resistant genes were tested, including erythromycin ribosomal methylase type A (ermA), type C (ermC), MAC-streptogramin resistance (msrA), to identify the macrolide resistance genes that could be involved in MAC resistance [14]. The antibiotic-resistant penicillin-binding protein PBP 2a, mecA, was tested to confirm MRSA phenotype, however, the specific thermo-nuclease (nuc) as a virulence gene was tested to confirm the pathogenicity and identity of the recovered S. aureus [27]. For the MDR S. aureus clinical isolates recovered in this study, results showed that all the detected MDR isolates (100%) were resistant to cefoxitin resistance, ampicillin, and amoxicillin-clavulanate, erythromycin, and clarithromycin. In addition to that, 80%, 74.6%, and 46.2% exhibited non-susceptibility to clindamycin, azithromycin, and PST, respectively. The lowest resistance was observed for linezolid (11.9%), followed by vancomycin (14.9%), teicoplanin (14; 14.9%), chloramphenicol (28.4%), and doxycycline (32.8%). According to the MDR S. aureus MAR index results (n = 67), each isolate had a unique MaR pattern, with MaR ranging from 0.31 to 1.0. The findings showed that two isolates (90.9%) had pan-drug-resistance (PDR) phenotypes due to MAR = 1.0, while 17 isolates (25.3%) had MaR indexes between 0.81 and 9.5, 41 isolates (61.2%) had MaR ranges between 0.5–0.77, and 7 isolates (10.4%) had MaR ranges between 0.31 and 0.45. The respective findings indicated a high level of antimicrobial resistance of the MDR isolates as compared to previous reports [38,39,40]. Therefore, further molecular analysis of the major antibiotic resistance genes was important to correlate antibiotic resistance to the potentially identified genetic determinants responsible for such resistance.
Molecular characterization revealed that all the MDR isolates (n = 67) gave 100% positive PCR for the nuc gene and mecA gene confirming that all isolates exhibited MRSA phenotypes. Regarding the MAC-resistant genes, the ermC (49.25%) was the most prevalent MAC-resistant gene, followed by the ermA (26.8%) and the msrA gene (23.8%). Our findings are in accordance with previous studies regarding the prevalence of the respective MAC-resistant genes among S. aureus clinical isolates [14, 35,36,37].
These findings initiated a further investigation to evaluate five antibiotic combinations of PST with each of the antimicrobial agents, FOX, LNZ, LEV, CN, and DO. The rationale for using these combinations was: i) PST is not commonly used in our clinical setting, so we expected to retain a better activity; ii) antimicrobial agents that still retain activity against MDR S. aureus based on our findings; iii) agents that are less expensive and commercially available in our clinical setting; and iv) those still not being evaluated. The synergistic/additive activities of certain combinations could be achieved regardless of whether the isolate is resistant to one of the tested antibiotics since it was based on the calculation of the ΣFIC index, which is based on the values of the MIC of each antibiotic alone and in combination. Our Findings showed that the studied MDR S. aureus (n = 67) exhibited 82.13% and 70.14% synergism with pristinamycin-doxycycline (PST-DO) and pristinamycin-levofloxacin (PST-LEV), respectively. However, they exhibited primarily additive effects (67%) when pristinamycin (PST) and linezolid (LNZ) were combined. In 71.6% and 52.2% of the tested MDR S. aureus isolates, respectively, pristinamycin-cefoxitin (PST-FOX) and pristinamycin-gentamicin (PST-CN) exhibited largely indifference.
Our study was focused on the use of PST as a new member of streptogramins and evaluated it either alone or as a combination of the other five antibiotics that are usually employed for the treatment of various infections caused by S. aureus. This is due to streptogramins’quick bactericidal action against a variety of pathogens, low incidence of resistance in clinical isolates, and favorable pharmacokinetic characteristics, which have made them viable substitutes for the treatment of Gram-positive infections [41]. PST looks to be a well-tolerated, effective oral option for treating tough Gram-positive infections, such as VRE and MRSA, in a variety of difficult-to-treat or protected-site illnesses [42]. Streptogramins, particularly PST, are viable and effective alternatives for the treatment of Gram-positive infections [41]. Oral treatment options for MDR S. aureus infections are limited. Pristinamycin (PST) is a possible alternative and has promising efficacy either alone or in combination with other antibiotics [43]. It was reported by Cocito et al. that MLSB phenotype alone is not sufficient to generate PST resistance as a variety of other resistance mechanisms is necessary to achieve high-level resistance to PST [44]. Despite 55% of isolates being resistant to lincosamides or having inducible resistance, Dancer et al. also found an 87% success rate in treating MRSA infections with PST, primarily affecting the skin and soft tissues, suggesting MLSB phenotype [45]. Similarly, Reid et al. treated 26 patients with primarily osteoarticular infections caused by 31 different staphylococci with PST. They found that eight were cured, 15 were suppressed, and 3 failed therapies because of drug intolerance, not uncontrolled illness [42]. Moreover, the in vitro MLSB phenotype of macrolide and lincosamide resistance does not appear to be related to PST resistance. Similarly, clinical investigations suggest satisfactory outcomes using PST in suspected MLSB staphylococcal infections [44]. Our findings showed that PST is the MAC antibiotic that exhibited the highest activity against MDR S. aureus. In addition, PST combination with doxycycline or levofloxacin achieved the highest synergism. These antibiotic combinations as highly recommended for clinical evaluation for potential use in humans against McRSA and MRSA clinical isolates.