Olive Leaf Extract as a Promising Topical Antimicrobial Agent Against Staphylococcus aureus and Its Resistant Strains, Including MRSA

1. Introduction

Staphylococcus aureus is naturally present on skin, nares and mucous membranes of healthy population, however it is responsible for more than one million deaths worldwide, being the leading bacterial cause of death in 135 countries [1]. Its great ability to adapt to the stressors present in the skin leads the bacterium to cause persistent and recurrent infections [2], and has been proven to be a disease promoter in inflammatory skin dermatoses as prevalent as atopic dermatitis and psoriasis [3,4]. Thus, it is one of the most frequently identified bacteria in skin and soft tissue infections (SSTIs) [5]. SSTIs range from mild infections, such as pyoderma, which are usually treated with oral antibiotics, to serious, life-threatening infections, such as necrotising fasciitis, which require intravenous treatment [6]. In SSTIs, S. aureus is responsible for more than thirty thousand infection-related deaths worldwide [1].

In this sense, the intensive use of antibiotics in recent decades has led to an increase in multidrug-resistant strains. In 2003, methicillin-resistant S. aureus (MRSA) was the major pathogen in SSTIs in patients from emergency department in the California region [7], as well as MRSA USA 300 clone was the main source of community-onset S. aureus SSTIs in Atlanta [8]. In 2019, in Europe more than 500,000 deaths were associated with bacterial antimicrobial resistance (AMR), with MRSA accounting for approximately 10% [9]. Nowadays, MRSA is responsible for more than 100,000 deaths worldwide and is one of the three antibiotic-resistant bacteria that cause the greatest burden of disease [5,10]. Hence, the rapid increase of antimicrobial-resistant bacteria leads to the challenge of obtaining new antibiotics and therapeutics, making it necessary to find alternative and efficient methods to treat skin infections. Otherwise, infection with antibiotic-resistant strains would be the main cause of death by 2050, including billions of dollars in costs to the population [11].

Traditionally, olive tree has been broadly used in medicine [12]. In particular, olive leaf extract (OLE) has been reported to have multiple uses as an hypotensive [13,14], antioxidant [15] and hypoglycaemic [16], and its polyphenols have also been associated with the reduction of proliferation of pancreatic cancer cells and the induction of apoptosis [17]. More specifically, OLE has shown antimicrobial in vitro activity against a diverse range of Gram-positive, Gram-negative bacteria and fungi (Candida albicans, Escherichia coli, dermatophytes, Helicobacter pylori, methicillin-sensitive S. aureus (MSSA), MRSA, anaerobic periodontal pathogens) [18,19,20,21]. Its antimicrobial properties have been attributed to polyphenols, particularly hydroxytyrosol and oleuropein [22,23]. OLE has been applied as an antimicrobial in food industry to control S. aureus growth during storage of Kasar cheese [24], to preserve seafood [25], to inhibit foodborne pathogens (Listeria monocytogenes, E. coli and Salmonella enteritidis) and avoid their biofilm formation [26].

Apart from that, olive tree products have been applied to the skin as anti-ageing, photo protective, anti-inflammatory and antioxidant agents, as well as for skin wound regeneration [27,28]. In mice, oral administration of OLE and oleuropein prevented the UVB-induced skin damage [29] and intradermal injections of oleuropein accelerated skin wound healing [30]. In rats, the combination of OLE with shea butter has been proven to be effective to treat non-diabetic and diabetic wounds when infected with MRSA [31]. In humans, topical application of olive oil improved the healing of diabetic foot ulcers [32]. Furthermore, it has also been tested in clinical trials for the treatment of papillomavirus and herpes simplex [33,34], achieving faster healing for both viruses infections.

Here, the present work faces two different main objectives. Firstly, to test a new olive leaf extract against S. aureus and its resistance form MRSA in order to combat skin and soft tissue infections, using both standard and clinical strains. Secondly, to epidemiologically describe the S. aureus strains isolated from SSTIs in the Microbiology Department of the Miguel Servet University Hospital over a three-month period, to characterise the number and type of resistances found in the clinical samples where OLE was tested. Hence, define the most common type of patients and samples that could benefit from the potential use of OLE as a treatment.

2. Results

2.1. Epidemiological Description of Clinical Samples

A total of 126 clinical samples were collected and analysed (Table S1). The clinical characteristics are summarized in Table 1.

The analysis of all samples revealed that the median patient age was 56 years (IQR: 27.5-73.8). Seventy-four samples were from male patients and 52 from female patients. Gender showed no association to samples regarding type of sample, location, associated pathology, age and number of antibiotic resistances. The majority of samples came from patients with skin infection without any previous dermatological disease (31%), followed by surgical procedure samples (22.2%). They were collected predominantly from skin lesions (42.9%) and were located on the trunk (22.2%), leg (23.0%), foot (19.0%) and head (19.0%) (Table 1). Thirty-three percent of samples from patients with surgical procedure (9/27) were associated with breast cancer (Table S1). Similarly, impetigo was the most prevalent condition among samples from patients with dermatological disease, occurring for 15 out of 23 cases (65.22%). Patients with skin infection who also had a dermatological disease were significantly younger (median age: 12.0 years, IQR: 6.5-18.0) compared to those with other associated pathologies (median age range: 47.0-79.0 years) (p value <0.05) (Table 1, Figure 1A). Similarly, samples from skin lesions were also obtained from younger patients (median age: 31.0 years, IQR: 12.3-54.5), compared to samples from surgical wounds (median age: 62.0 years, IQR: 47.8-72.5) or other ulcers (median age: 73.0 years, IQR: 64.5-86.8) (p value = 0.011 and <0.001, respectively) (Table 1, Figure 1B).

2.2. Antibiotic Resistance Patterns

Of the 126 samples, 103 (82%) were identified as MSSA and 23 (18%) as MRSA, 63 (61%) and 11 (48%) from male patients respectively. Over 90% of MSSA strains showed less than five antibiotic resistances, whereas 82% of MRSA strains were resistant to more than six antibiotics (Table 1). As expected, the number of antibiotic resistances was statistically higher in MRSA than in MSSA (Figure 1C). In addition, a positive correlation was found between patient age and the number of antibiotic resistances in MRSA strains, the older the patient, the higher the number of antibiotic resistances (Figure 2). With regard to the overall quantity of antibiotic resistances, strains isolated from pressure ulcers showed higher resistance levels compared to those from skin lesions (p value= 0.034) and surgical wounds (p value = 0.039) (Table 1, Figure 1D). It is noteworthy that all the strains from abscesses, surgical wounds, skin lesions, and other ulcers exhibited less than five antibiotic resistances, whereas 60% of pressure ulcer isolates had more than 11 antibiotic resistances (median value: 11.0, IQR: 6.0-13.0) (p value <0.001) (Table 1, Figure 3). Furthermore, a higher prevalence of MRSA was noted in these isolates compared to the rest (p value <0.05) (Figure 4).

2.2.1. Susceptibility Tests on ATCC (American Type Culture Collection) Samples

The susceptibility test on agar plate showed an inhibition of growth in S. aureus at 25, 12.5 and 6.25%w/v, whereas in MRSA OLE was effective at 25, 12.5, 6.25 and 3.125% (w/v). The results of the broth method dilution demonstrated that the OLE MBC for MSSA ranged from 3.125 to 6.25% and for MRSA from 1.56 to 3.125% (w/v), depending on the extraction batch and time since extraction. At least three different extraction batches were analysed. Notwithstanding the observed variation, all samples were effective at a concentration of 6.25% of OLE.

Regarding the lethal curve, the concentration of OLE employed was the maximum feasible (12.5% w/v), aiming for rapid bactericidal activity. After two hours, a reduction of 6 log₁₀CFU/ml was achieved in both MSSA ATCC 29213 and MRSA ATCC 700699 strains treated with OLE against control group (p value <0.005) (Table 2, Figure 5). No bacterial growth (neither MSSA nor MRSA) was observed after 24 hours of OLE treatment, whereas both strains reached a bacterial load of 10⁹ CFU/ml when water was used as control treatment (Table 1, Figure 5).

2.2.2. Susceptibility Tests on ATCC Samples

All clinical isolates described were susceptible to OLE at concentrations of 25, 12.5 and 6.25% (w/v), with seven strains susceptible at 3.125% w/v (Table S1). All the strains were susceptible to seven antibiotics, namely ceftaroline, linezolid, daptomycin, pristinamycin, trimethoprim-sulfamethoxazole, teicoplanin and vancomycin. Conversely, penicillin and ampicillin exhibited the highest resistance frequency (Table 3, Figure 6); in fact, all MRSA strains were resistant to any penicillin (Table 3). Resistant to all aminoglycosides, all penicillins, cefdinir, ciprofloxacin, levofloxacin, erythromycin, quinupristin-dalfopristin, minocycline, fosfomycin, mupirocin and rifampicin was significantly higher in MRSA strains compared to MSSA (p <0.05) (Table 3). Fluoroquinolone-resistant strains were also resistant to moxifloxacin (Table S1).

The interpretation of fusidic acid susceptibility was limited due to testing panel concentrations (2 mg/l) that did not align with EUCAST breakpoints (1 mg/l and 0.5 mg/l for topical use) (The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 14.0, 2024. http://www.eucast.org). Subsequently, an additional antimicrobial disk susceptibility test was conducted based on clinical characteristics and physician requests at the Microbiology department. Apart from that, up to 60% of MRSA analysed isolates were resistant to fusidic acid (Table 3).

When looking for more than one antibiotic resistance in each sample, 8 out of 126 strains were resistant to oxacillin and gentamicin. Additionally, six (three MSSA, three MRSA) out of 28 strains analysed were resistant to both fusidic acid and mupirocin. Similarly, three MRSA out of 28 strains tested were resistant to both fusidic acid and gentamicin. Furthermore, two of these MRSA were resistant to the four antibiotics fusidic acid, gentamicin, mupirocin and oxacillin (Table S1).

2.2.3. Antibiotic Families and Efficacy

In terms of antibiotic susceptibility by antibiotic family, penicillins had the lowest susceptibility rate (19.8%) (Figure 6), while oxazolidinones (linezolid), lipopeptides (daptomycin), pristinamycin, folate pathway inhibitors (trimethoprim-sulfamethoxazole), glycopeptides and lipoglycopeptides (teicoplanin and vancomycin), and OLE showed 100% efficacy (Figure 6).

3. Discussion

The present study demonstrated the antibiotic effect of OLE against multidrug-resistant strains of S. aureus, including the determination of their MBC and lethality curve, based on both reference and hospital clinical samples. Additionally, it provides and epidemiological description of the clinical samples analysed, hence, defining the profile of patient that could benefit from the treatment with OLE.

In our research, susceptibility test on agar plate and MBC values were slightly lower in the MRSA ATCC strain when compared to the MSSA ATCC strain. This variation can be attributed to the faster in vitro growth rate of MSSA, which is characterised by its metabolic efficiency and adaptability. It has been reported that MSSA strains generally exhibit faster replication rates under laboratory conditions, whereas MRSA mechanisms of resistance frequently incur a metabolic cost [35,36]. Furthermore, the genetic diversity among MRSA strains can also contribute to variability in growth rates, as demonstrated by Stevens et al., who showed differences in growth speed and antibiotic tolerance in a MRSA strain [37].

Regarding other studies on OLE, Sujana et al. reported a minimum inhibitory concentration (MIC) of 0.62% for S. aureus with a minimum oleuropein content of 4.4 mg/ml [20]. In our case, the amount of oleuropein was 8.3 mg/ml (Table S2) and the MBC were slightly higher, being 6.25% for MSSA and 3.125% for MRSA. This difference may be attributed to the extraction method, since Sudjana et al. employed fresh leaves whereas we used powdered dried leaves. Markin et al. reported a MBC of 0.6% w/v for S. aureus from powdered leaves, but they stored their extract for up to eight weeks [19], while our OLE was used after being frozen at -80°C for six months (data not shown). Apart from that, the majority of studies report the antimicrobial effect of OLE after a 24-hour incubation period [20,21,26]. In our research, the OLE was able to reduce the number of viable bacteria to nearly zero after two hours of incubation, consistent with the findings of Markin et al. [19], but we also tested a MRSA ATCC strain.

Most authors assign the antimicrobial activity of OLE to polyphenols, such as oleuropein and its derivatives [22,23,38,39]. The amount of oleuropein is commonly used as a standard of OLE characterization [20]. On the other hand, oleanolic acid has been shown to cause cell membrane damage in L. monocytogenes, Enterococcus faecium, and Enterococcus faecalis [40]. Olive leaf extract has also been reported to suppress flagella production in L. monocytogenes [26] and polyphenols from olive mill wastes showed inhibition of E. coli biofilm formation [41]. Although the mode of action of OLE is not fully understood, its antimicrobial efficacy is believed to stem from the synergistic effects of its constituent compounds [23]. Liu et al. reported greater inhibition of bacterial growth when using the olive leaf extract compared to oleuropein and verbascoside separately [26]. Furthermore, Lim et al., (2016) showed enhanced ampicillin efficacy when combined with OLE, highlighting its potential to mitigate antibiotic resistance development through a multi-compound mechanism [43]. In our study, the efficacy of OLE was evaluated against a range of clinical strains of S. aureus, including those exhibiting diverse antibiotic resistance profiles. Even the most resistant MRSA strains were susceptible to OLE at three different concentrations (Table S1).

Regarding skin and soft tissue infections, in our study 31% of the samples derived from a skin infection without any underlying dermatological disease, such as burns, wound infections, trauma, etc. (Table 1). Similarly to other researchers that identified impetigo as the most predominant infection [44], in our case it accounts for the 65% of samples collected from patients with a dermatological disease (Table S1). The frequency of MRSA found in skin and soft tissue infections varied between studies. In our research there were 18.3% MRSA (Table 1); lower than the 54.1% reported in Pakistan [44], and also slightly lower than the Spanish prevalence of 20-30% reported by Murray et al. [9]. However, a higher proportion of MRSA (66.6%) was found in pressure ulcer samples (Figure 3), similar to the 59% reported by Manzur et al. [45].

The most common treatment for S. aureus infection involves oral antibiotic therapy or parenteral treatment for severe cases [5,46] as well as for MRSA infection, including a wide range of adverse reactions [47]. On the other hand, the most commonly used topical antibiotics are fusidic acid or mupirocin [48]. Frazee et al. (2005) proposed the use of trimethoprim/sulfamethoxazole, clindamycin or vancomycin for the treatment of skin and soft tissue infections in communities with high MRSA prevalence [7]. In contrast, Habib and Qadir (2022) recommended rifampicin, minocycline, amikacin, and clindamycin for MRSA, reserving linezolid, teicoplanin and vancomycin for severe cases [44]. We reported only seven antibiotics, apart from OLE, demonstrating susceptibility across all strains (ceftaroline, linezolid, daptomycin, pristinamycin, trimethoprim-sulfamethoxazole, teicoplanin and vancomycin) (Table 3). In comparison to previous studies [7,44,49,50], the data presented here showed higher rates of antibiotic resistance, especially to fluoroquinolones, clindamycin, gentamicin, fusidic acid and mupirocin. In our research, MSSA showed a higher resistance rates for clindamycin (27.2%), fusidic acid (22.2%) and mupirocin (12.6%) in comparison to the 8.8%, 10.9% and 1.4% respectively reported by Zhanel et al. (2021). Similarly, the MRSA resistance rates for levofloxacin (78.9%), ciprofloxacin (73.9%), clindamycin (47.8%), fusidic acid (60%) and mupirocin (39.1%) were higher than the 43.2% reported by Frazee et al. (2005) for levofloxacin or the 67.1%, 9.5%, 10.6% and 14.1% reported by Zhanel et al. (2021) for ciprofloxacin, clindamycin, fusidic acid, and mupirocin respectively [7,50] (Table 3). Although the susceptibility rate to fusidic acid in our research may be overestimated, as only 28 strains were analysed due to the limitations of the technique.

Looking in detail at topical antibiotic resistance, up to 22% of samples analysed were resistant to both fusidic acid and mupirocin simultaneously, which could lead to failure of topical treatment and progression to more severe disease. Additionally, two strains analysed were found to be resistant to fusidic acid, gentamicin, mupirocin and oxacillin, whereas all these strains reported were susceptible to OLE (Table S1).

Besides, OLE and its derivatives have already been successfully applied on the skin as a protective and antioxidant factor, to reduce skin damage, and as a healer of diabetic foot ulcers [27,28,29,30]. Moreover, it has even been employed in the treatment of papillomavirus and herpes simplex virus infections [33,34]. For these reasons, OLE may be used not only to treat but also to prevent skin infections caused by S. aureus. For example, 33% of our samples from surgical procedures were from breast cancer wounds. Given that OLE is known to help with skin regeneration [27], it could be used after surgery to prevent the infection and improve wound healing. In a similar way, it could benefit patients with atopic dermatitis by helping to control the disease by reducing the amount of S. aureus in their microbiome while promoting skin barrier repair [3]. In addition, the application of OLE could be useful in patients with comorbidities, where parenteral administration of antibiotics is ineffective due to the difficulty of reaching the local niche of the bacteria. It could also be applied in patients with antibiotic restrictions, such as children or pregnant women. In our research, the median age of patients with a skin infection and dermatological disease was 12 years old (Table 1), of which 65% were impetigo cases. These patients could benefit from treatment with OLE, thus reducing the spread of this highly contagious paediatric condition.

The potential of OLE as a topical antimicrobial is highlighted by the present work. We propose a localized treatment with OLE as single agent or in combination with other antibiotics to prevent the dispersion of bacteria, given that OLE is already sold as a natural supplement to reduce high blood pressure and decrease cholesterol levels among others, making it easy to implement in the clinic. For instance, it could be useful in countries with high MRSA prevalence [9], taking advantage of the lack of side effects and cost-effective treatment. However, further research is needed to know more about its mechanism of action, stability, skin penetration level and efficacy, alone or in combination with other antimicrobials, in clinical trials involving patients with skin and soft tissue infections.

To our knowledge, this is the best time that OLE is proposed as a topical bactericide to fight against dermatological infections. Furthermore, this is the first report demonstrating that its antimicrobial action, after 2 hours incubation, leads to a complete bacterial eradication of MSSA and MRSA. Additionally, it has been tested on different strains of S. aureus, with varying levels of antibiotic resistances. Moreover, a clinical description of all the S. aureus strains isolated from patients with skin and soft tissue infections in our department was conducted, identifying the most common antibiotic resistance patterns and defining the patient profile that could benefit from the potential use of OLE as a treatment.

4. Materials and Methods

4.1. OLE Extraction

Olive leaf extract was prepared as follows: dried olive leaves of Olea Europaea, stored in an opaque container under nitrogen atmosphere, were weighed on a precision balance and diluted in bidistilled and sterile water at room temperature to a concentration of 25% weight/volume (250 mg/ml). Once the dilution was prepared, it was homogenised with two zirconium/silica beads in a Mini Beadbeater 16 cell disruptor (BioSpec®) at 3450 rpm for 2 minutes. The extract was then subjected to an ultrasound bath for 3 minutes at 37°C. The supernatant was separated from the leaf debris by centrifugation at 5000 rpm for 2 minutes. The aqueous phases of all the fractions were pooled and centrifuged again. Finally, the olive leaf extract was filtered through a 0.22 µm filter and stored in sterile Eppendorf tubes. Once the extract was obtained, the aliquots were kept frozen at -80°C until use.

4.2. OLE phenolic Profile

OLE extract was homogenised with 70% ethanol in Milli-Q water (1,600 rpm, 2 min) using an Ultra-Turrax T25-Basic mixer. Then, the mixture was centrifuged (12,500 rpm, 4°C, 15 min) and the supernatant was filtered through a 0.45 μm membrane. The resulting extracts were stored at -18°C in the dark until further analysis. Identification and quantification of the individual phenolic compounds were carried out by UPLC-MS/MS on an AcQuity Ultra-Performance™ liquid chromatography/tandem mass spectrometry system (Waters, Milford, MA, USA) similarly to that described by Martínez-Beamonte [51]. Calibration curves with an R² value of 0.99 or higher were generated using commercial standards (Sigma-Aldrich, Madrid, Spain) to ensure accurate quantification. The phenolic profile of OLE is presented in Table S2.

4.3. Bacteria

4.3.1. Reference S. aureus Strains

The reference strains used were MSSA American Type Culture Collection (ATCC) 29213 and MRSA ATCC 700699 and cultured on Columbia blood agar (CBA) (Thermo Scientific™) under aerobic conditions at 35°C for 18-24 hours to obtain fresh isolated colonies. These strains were used to test the bacterial susceptibility to OLE on agar plate, to calculate the minimum bactericidal concentration (MBC), and the lethal curve.

4.3.2. S. aureus Strains from Clinical Samples

This study included all samples from patients with skin and soft tissue infections that were received between May 29th and August 29th 2024 at the Microbiology Department of Miguel Servet University Hospital (the catchment area covers approximately 400,000 people), in which S. aureus was isolated. Identification was performed by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) (Bruker Daltronik GmbH®). These isolated strains were used to test the bacterial susceptibility to OLE on agar plate, and to stablish its antibiotic susceptibility profile.

Data related to antibiotic susceptibility, sample type, location of infection, associated pathology (when possible) and patient age and gender was collected for each sample. Type of sample was classified according to standard laboratory practice as abscess, skin lesion, surgical wound, ulcer and pressure ulcer. Location was categorized as well as: generalized all body, head, hand, arm, trunk, leg, foot and toenail. The associated pathology was divided into six groups as follows: a) immunosuppression (samples associated with angiosarcoma, basal cell carcinoma and squamous cell cancer); b) surgical procedure (samples from catheter, surgery, amputation and prostheses); c) skin infection without dermatological factors (burns, insect stings, omphalitis, knife cuts, trauma and any skin and wound infection); d) skin infection with dermatological disease (epidermolysis bullosa, herpes zoster, impetigo, psoriasis and scalded skin syndrome); e) vascular disease (elephantiasis, lymphedema, lymphocele and vascular ulcer); f) pressure ulcer (immobilization); and g) diabetic foot.

4.4. OLE Bacterial Susceptibility Test on Agar Plate

The inoculum was prepared by direct saline suspension of fresh isolated colonies and adjusting the turbidity to 0.5 McFarland in a MicroScan turbidity meter (Beckman Coulter®), thus containing approximately 1-2x10⁸ CFU/ml (colony-forming units). A sterile cotton swab was placed in the bacterial suspension and streaked in three directions over the surface of the Müller-Hinton agar (MHA) (Oxoid™) to obtain uniform growth. Then, 10 µl of the OLE was added on the surface, as well as sterile water as negative control. The concentrations of OLE tested were 25% w/v, 12.5% w/v, 6.25% w/v and 3.125% w/v. Plates were incubated at 35°C for 24 hours [52]. The categorisation of strains as either susceptible or resistant was determined by the presence or absence of growth. For the clinical samples, at least two different aliquots of OLE were tested.

4.5. Minimum Bactericidal Concentration (MBC)

The MBC of OLE was determined for the two reference strains by broth microdilution method according to the Clinical Laboratory Standards Institute guidelines [53] with little variation. An inoculum of 0.5 McFarland was prepared as described above. The inoculum was diluted in Cation Adjusted Müller-Hinton Broth (CAMHB) (Becton Dickinson®) to achieve a concentration of 1x10⁶ CFU/ml. 50 µl of the inoculum was then incubated with 50 µl of serial dilutions of OLE to a final concentration of approximately 5x10⁵ CFU/ml per tube. The final concentrations of OLE tested were 12.5, 6.25, 3.12, 1.56 and 0.78% (w/v). The tubes were incubated for 18-20 hours at 35°C. After that, the 100 µl were spread on CBA plates and incubated again 24 hours. The MBC was the dilution at which no growth was observed in each strain. Three different batches of OLE were tested.

4.6. Lethal Curve

The protocol was similar to that described for the MBC. In this case, 50 µl of the inoculum were mixed with either 50 µl of 25% w/v OLE or 50 µl of sterile water (negative control) and incubated at 35°C for 2, 4, 8 and 24 hours. Subsequently, the entire contents of the tubes were spread on CBA plates and the colonies were counted after 24-hour incubation period. Regarding the inoculum, 10 µl of a 1/10000 dilution of the inoculum was spread over the surface of a blood agar plate, the presence of approximately 100 colonies indicated an initial inoculum density of 1x10⁸ CFU/ml, thus 5x10⁵CFU/ml in each assay. Additionally, the negative control for each time point was also diluted and 10 µl used for the colony counting. A minimum of six replicates, comprising two different extract batches, were conducted for each strain.

4.7. Antibiotic Susceptibility

Antibiotic susceptibility testing was done by Microscan Walkway™ semi-automatic system (Beckman Coulter®), Pos MIC panel type 33 with the following antibiotics and dilutions (µg/ml) ranges according to the manufacturer (https://www.beckmancoulter.com/es/products/microbiology/conventional-panels#/): amikacin (8-32), amoxicillin/clavulanic acid (4/2-8/4), ampicillin (0.25, 4-8), ceftaroline (0.5-1), chloramphenicol (8-16), ciprofloxacin (1-2), clindamycin (0.25-2), daptomycin (1-4), erythromycin (0.5-4), fosfomycin (32-64), fusidic acid (2), gentamicin (1-8), imipenem (4-8), levofloxacin (1-4), linezolid (1-4), minocycline (1-8), moxifloxacin (0.5-1), mupirocin (256), nitrofurantoin (32-64), norfloxacin (4-8), oxacillin (0.25-2), penicillin (0.12-0.25, 8), pristinamycin (1-2), rifampin (0.5-2), synercid (quinupristin-dalfopristin) (1-4), teicoplanin (1-16), tetracycline (1-8), tobramycin (1-8), trimethoprim/sulfamethoxazole (2/38-4/76) and vancomycin (0.25-16). For some samples only disk diffusion susceptibility test was done, using disks of: fusidic acid (10 µg), cefoxitin (30 µg), ciprofloxacin (5 µg), clindamycin (2 µg), erythromycin (15 µg), gentamicin (10 µg), linezolid (10 µg), mupirocin (200 µg), rifampicin (5 µg), tetracycline (30 µg), oxacillin (1 µg) and trimethoprim-sulfamethoxazole (25 µg). According to EUCAST [54] the antimicrobial susceptibility was categorized as S (susceptible), I (susceptible, increased exposure) and R (resistant) for each antibiotic tested.

4.8. Statistical Analysis

The Values were expressed as mean and standard deviation (SD) or median value (P25-P75). Normality was tested by Shapiro-Wilk. Differences between the experimental groups in terms of number of colony-forming units were assessed using Students’ t- or Mann-Whitney U test depending on the normality. Differences in clinical samples regarding sample type, pathology and location with number of antibiotic resistances and age were analysed using Kruskal-Wallis test. The association between age and number of antibiotic resistances was evaluated with Pearson correlation coefficient. Two-tailed p value was considered statistically significant when p < 0.05. All analyses and graphs were carried out with the free based R software Jamovi 2.2.5 [55], MS Excel 2016 (Microsoft, WA, USA), MS PowerPoint 2016 (Microsoft, WA, USA) and MS Word 2016 (Microsoft, WA, USA). Images from visual abstract were downloaded from freepik (https://www.freepik.es/) and pixabay (https://pixabay.com/es/).