Introduction
Skin and soft tissue infections (SSTIs) are prevalent and may be serious, hospitalizing 7-10% of patients globally. 1 Many topical medications are available to treat such infections having localized antibacterial action and fewer systemic side effects. They are more patient-compliant with ease of application and storage. For systemic skin Mupirocin, Clindamycin, and Fusidic acid are administered topically. However, bacterial resistance to these medications has increased, prompting the development of new broad-spectrum antibiotics with reduced antimicrobial resistance.2 Nadifloxacin is another viable option for acne and other bacterial skin infections. Its antibiotic action targets aerobic gram-negative, gram-positive, and anaerobic bacteria. Skin infections may benefit from Nadifloxacin's broad-spectrum antibiotic action. In situations of topical agent resistance, it offers an alternate therapy. Healthcare practitioners may successfully treat bacterial infections while avoiding antimicrobial resistance with this medication.3, 4
Previous in vitro research on bacterial skin infections showed that Nadifloxacin is safe and effective against a range of bacteria. It's very effective against Streptococcus and Propionibacterium species. These data showed that Nadifloxacin may cure bacterial skin infections by targeting a wide spectrum of pathogens.5 Nadifloxacin had antibacterial action against S. epidermidis, P. acnes, MSSA, and MRSA, and none of these pathogens were resistant to Nadifloxacin, demonstrating its efficiency in reducing their growth.6, 7 The present study examines the antimicrobial susceptibility of few gram-positive and gram-negative organisms (S. aureus, S. pyogenes, S. epidermidis, methicillin-resistant S. epidermidis, methicillin-resistant S. Aureus E. faecalis, P. aeruginosa, and P. acne) to four topical antibiotics: Mupirocin, Clindamycin, Fusidic acid, and Nadifloxacin.
Methodology
Antimicrobial susceptibility testing (AST) on aerobic and anaerobic bacteria was done using the Kirby-Bauer disk diffusion technique. The study utilized 76 isolates of different organisms. This standard method is often used to test quickly developing bacteria' antibiotic sensitivity and resistance. Most organisms grew on Mueller Hinton agar, except P. acne, which grew on Brucella blood agar. After overnight incubation, filter paper disks impregnated with calibrated doses of antimicrobial agents were tested for Zone of Inhibition (ZOI) size. Incubation periods were specified by the Clinical and Laboratory Standards Institute (CLSI) performance standards for antimicrobial disk susceptibility tests. The testing included quality control strains P. aeruginosa ATCC 27853 and S. aureus ATCC 25923. Epsilometer test (E-Test) and Micro-broth dilution were used to evaluate minimum inhibitory concentration (MIC) values in the research. Micro-broth dilution process entailed loading microtiter plates with broth and putting two-fold antibiotic dilutions into the wells. And dispensing bacterial isolates into the respective wells. The plates were incubated for 16–20 hours, and then they were visually examined to see whether the bacteria had grown. The Clinical and Laboratory Standard Institute investigated the antibiotic resistance profile of Staphylococcus species, including S. aureus and S. epidermidis isolates and fresh isolates.
Using the HiComb approach, individual bacterial strains' susceptibility or resistance was quantified. Dry chemistry and a gradient-based method were used. The apparatus included two comb-shaped strips with extensions that held antibiotic-loaded discs. On an agar plate, the discs generated a concentration gradient of the antibiotic through 16 two-fold dilutions. An oval ZOI formed on the agar surface as the antibiotic diffused from one end of the strip to the other. Where the zone met the strips' comb-like projections, the MIC was calculated. The CLSI process and this method's MIC are comparable. HiComb strips from HiMedia Laboratory Ltd. were used to cultivate diverse organisms on Muellar Hinton or Brucella blood agar. According to the manufacturer, the ZOI was tested and reported as sensitive or resistant. The testing also included quality control strains P. aeruginosa ATCC 27853 and S. aureus ATCC 25923. The concentration of antibiotics used in the experiment included 200 μg/ml of Mupirocin, 2 μg/mL of Clindamycin, 10 μg/mL of Fusidic acid, and 5 μg/ml of Nadifloxacin. The efficacy of Nadifloxacin was evaluated for fresh 25 Staphylococcus sp (S. aureus, S. pyogenes, S. epidermidis, methicillin-resistant S. epidermidis, S. aureus) and E. faecalis, P. aeruginosa, and P. acne isolates using the same protocol and compared against 200 mcg Mupirocin. The approach also evaluated ZOI & MIC of Nadifloxacin versus Mupirocin, Clindamycin, and Fusidic acid for different bacterial strains of MRSA, P. acnes, and S. epidermidis, which cause SSTIs.
Results
Disk diffusion
Table 1 depicts the findings obtained for antibiotics on the tested microorganisms. All isolates of E. coli (SRL 7, SRL 43, SRL 44, SRL 45, SRL 66, SRL 68, SRL 69, SRL 70, SRL 71, SRL 72, SRL 73, SRL 74, SRL 75) were sensitive to Nadifloxacin with ZOI values ranging between 9 to 57. Fusidic acid showed a high level of resistance towards SRL 7 while being resistant towards other E.coli isolates. However, other E.coli isolates demonstrated resistance or no ZOI against Clindamycin and Fusidic acid.
The E. faecalis isolates showed the absence of high-level resistance against a Nadifloxacin reference standard, DRL API, and high media, along with Mupirocin while it showed resistance against Clindamycin. SRL 5,13,14,20,21,26,30,31,33,36,54, and 55 were sensitive against Fusidic acid while SRL 61 was resistant.
MRSA isolates SRL 1, SRL 10, SRL 12, SRL 15, SRL 23, SRL 24, SRL 27, SRL 29, SRL 64, and SRL 65 exhibited the absence of high level of resistance to Nadifloxacin (reference and API), Nadifloxacin, and Mupirocin while it exhibited sensitivity to Clindamycin and Fusidic acid. MRSA isolates exhibited ZOI in the range of 16 to 38 for all antibiotics.
Table 1
Antibiotic susceptibility testing results
Table 0
Table 0
Table 0
Table 0
Table 2
MIC values for various antibiotics against different bacterial isolates
MSSA isolates SRL 35, SRL 41, SRL 42, SRL 46, SRL 2, SRL 3, SRL 4, SRL 9, SRL 11, SRL 16 exhibited absence of high level of resistance to Nadifloxacin (reference and API), and Mupirocin while it exhibited sensitivity to Clindamycin and Fusidic acid except SRL 2 and SRL 3, which showed resistance to Clindamycin while SRL 4, 46, and 59 showed resistances to Fusidic acid. The ZOI exhibited was in the range of 14 to 41 for all antibiotics.
S. epidermidis isolates SRL 48, 56, 57, 58, and 49 were resistant to Clindamycin, and SRL 62, 63, and 40 were sensitive to it. Similarly, SRL 48, 56, 57, and 58 were resistant to Fusidic acid while SRL 62, 63, 40, and 49 were sensitive to it. S. epidermis isolates exhibited ZOI in the range of 11 to 41 for all antibiotics. SRL 62 and 49 show the absence of high-level resistance against Nadifloxacin reference and DRL API, while other S. epidermis isolates show the absence of high-level resistance against Nadifloxacin high media and Mupirocin.
S. pyogenes isolates SRL 60 showed absences of the high level of resistance to all antibiotics except Fusidic acid with ZOI ranging from 12 for Fusidic acid to 28 for Mupirocin. On the other hand, P. acnes exhibited an absence of high-level resistance towards Nadifloxacin high media Mupirocin, Clindamycin, and Fusidic acid.
MIC Results
Table 6 provides the MIC values for various antibiotics against different bacterial isolates. For Mupirocin, it was observed that 60% of gram-positive isolates showed a MIC value of less than 4 μg/ml, indicating moderate sensitivity. However,some gram-negative isolates exhibited a high level of resistance with MIC values exceeding 240 μg/ml. Clindamycin demonstrated moderate sensitivity against gram-positive isolates, with 58% of isolates showing a MIC value of less than 0.5 μg/ml. Among gram-negative isolates, 92% showed a MIC value greater than 60 μg/ml, indicating very high resistance. Fusidic acid exhibited the lowest MIC values (< 0.5 μg/ml) for 96% of gram-positive isolates, indicating strong efficacy against this group. However, all gram-negative isolates showed a MIC value greater than 240 μg/ml, indicating complete resistance. Nadifloxacin demonstrated a MIC value of less than 4 μg/ml for 70% of all isolates, indicating a high level of sensitivity. The results suggest that Mupirocin and Clindamycin have moderate effectiveness against gram-positive isolates but are less effective against gram-negative isolates. Fusidic acid shows excellent efficacy against gram-positive isolates but is ineffective against gram-negative isolates. Nadifloxacin demonstrated a high level of sensitivity across all isolates.
Disk Diffusion and MIC Results from the Extension Study
Among the 57 Staphylococcus species isolates, including 49 isolates of S. aureus and 8 isolates of S. epidermidis, the antibiotic susceptibility testing revealed that all isolates (100%) showed sensitivity to Nadifloxacin as determined by both the disk diffusion method and broth dilution method, with MIC value <4 μg/ml.
On the other hand, 95% of the isolates were sensitive to Mupirocin as determined by the disk diffusion method. However, when tested with the E-Test, a slightly lower sensitivity of 88% (MIC < 4 μg/ml) was observed, indicating sensitivity. Four isolates showed discordant results, with high MIC values but sensitivity observed by the disk diffusion method using a Mupirocin disk concentration of 200 μg. This discordance could be attributed to the presence of low-level resistance to Mupirocin.
The disk diffusion method showed 88% sensitivity to Fusidic acid. The MIC values for 89% of the isolates were low, below 1.0 μg/ml, and borderline (1.0 μg/ml) for the remaining 11% of isolates, indicating sensitivity. The correlation between the disk diffusion and broth MIC results was 79%. Among the 11 discordant isolates, four showed borderline MIC values, while six exhibited borderline zone sizes between 12-16 cm by the disk diffusion method. The correlation between the disk diffusion method and E-Test results was 96% for MIC values below 0.5 μg/ml. Two isolates showed discordant results, which could be attributed to procedural bias. Additionally, one isolate of S. epidermidis was found to be resistant to the tested antibiotics except Nadifloxacin, for which it was sensitive with a MIC value below 0.0625 μg/ml.
Regarding resistance rates, Clindamycin exhibited the highest resistance, with 12% of the isolates being resistant according to both the disk diffusion method and E-Test (MIC > 0.5 μg/ml). Fusidic acid showed resistance in 11% of the isolates according to the disk diffusion method, and 12% resistance according to the E-Test (MIC > 1 μg/ml). Mupirocin demonstrated a resistance rate of 5% based on the disk diffusion method and 12% based on the E-Test (MIC < 4 μg/ml). Although only 5% of methicillin-resistant isolates demonstrated high-level resistance to Mupirocin by disc diffusion technique, all these isolates were susceptible to Nadifloxacin, demonstrating superiority.
Overall, Nadifloxacin demonstrated excellent sensitivity, Mupirocin showed good sensitivity with some discordant results possibly due to low-level resistance, and Fusidic acid exhibited a high sensitivity rate with a few isolates showing borderline MIC values (Figure 1 -10 of Table 3 and (Figure 11, Figure 12 of Table 4).
Discussion
This study evaluated the antibiotic susceptibility patterns of common bacterial strains causing SSTIs. Specifically, the study aimed to assess the ZOI and MIC of four topical antibiotics- Nadifloxacin, Mupirocin, Clindamycin, and Fusidic acid against bacterial strains. Nadifloxacin was effective against 70% of the isolates at a MIC of < 4 μg/ml. The results of the study revealed that Nadifloxacin consistently exhibited a larger ZOI compared to the other antibiotics for all bacterial strains tested. This indicates that Nadifloxacin has a higher efficacy in inhibiting the growth of these bacterial strains, suggesting its potential as a first-line treatment option for SSTIs caused by these organisms. The larger ZOI can be attributed to Nadifloxacin's specific mechanism of action, targeting bacterial DNA gyrase and topoisomerase IV, which are crucial for bacterial replication.8 A similar study conducted by Alba et al., 2009 investigated Nadifloxacin against isolates of P. acnes, MSSA, MRSA, and S. epidermidis from Spain, Hungary, and Germany. 9 The study demonstrated that Nadifloxacin outperforms the comparators (Ciprofloxacin, Erythromycin, and Clindamycin) against the above-mentioned bacteria and has no additional effect on resistance.9
Nadifloxacin is found to be effective when used against both aerobic and anaerobic isolates. MIC90 values of dadifloxacin for S. aureus was 0.1 g/ml, Streptococcus spp. was 0.78 g/ml, and Propionibacterium spp.'s was 0.39 g/ml. Other antibiotics, however, showed resistance, with some strains having MICs higher than 12.5 g/ml. 7
Mupirocin, another topical antibiotic commonly used in the treatment of SSTIs, showed moderate activity against the bacterial strains tested. The ZOI observed for Mupirocin varied among the different strains, indicating a relatively lower efficacy compared to Nadifloxacin. However, it is important to note that Mupirocin is still considered effective against certain bacterial species causing SSTIs, particularly strains of S. aureus. Thus, its use may be warranted in cases where Nadifloxacin is contraindicated or when targeting specific bacterial species known to be susceptible to Mupirocin.10
Fusidic acid, an antibiotic used in SSTI management, displayed varying susceptibility patterns among the bacterial strains. Some strains showed a relatively large ZOI, indicating high susceptibility to Fusidic acid, while others demonstrated a smaller ZOI, suggesting reduced susceptibility. This finding suggests that the use of Fusidic acid as a monotherapy for SSTIs should be approached with caution, as its effectiveness may vary depending on the specific bacterial strain involved. Combination therapy or alternative treatment options may be employed in cases of reduced susceptibility to Fusidic acid.11, 12 The effectiveness of Mupirocin cream and topical Fusidic acid in treating experimental S. aureus infections was comparable, aligning with clinical observations. Nonetheless, Fusidic acid's effectiveness is reduced against streptococci and is particularly less efficient than Mupirocin cream in addressing S. pyogenes infected wounds.13
Clindamycin, a broad-spectrum antibiotic, exhibited varied susceptibility patterns across the bacterial strains tested. Some strains showed a significant ZOI, indicating high susceptibility, while others demonstrated reduced susceptibility. This suggests that the efficacy of Clindamycin against SSTIs may be dependent on the specific bacterial strain involved.10 The rates of Clindamycin resistance in MRSA were naturally greater than those in MSSA. Interestingly, just 4% were resistant to Nadifloxacin. The discrepancy may be because Nadifloxacin predominantly targets DNA gyrase.9
Previous research has extensively examined Nadifloxacin's bactericidal effects. It displays remarkable in vitro activity against both aerobic and anaerobic bacteria, including S. epidermidis, S. aureus, S. pyogenes, Streptococcus viridans, E. coli, P. acnes, and P. granulosum. 14, 15, 16, 17, 18, 19, 20, 21, 22 The MIC50 values were determined as 0.25 Ìg/ml for P. acnes, 0.125 Ìg/ml for P. granulosum, 0.03 Ìg/ml for S. aureus and 0.06 Ìg/ml for CNS. Notably, no resistance to the fluoroquinolone Nadifloxacin was detected, consistent with the findings of Kurokawa et al. 23 The current study's outcomes align with Vogt et al., 16 who similarly found no Nadifloxacin-resistant strains of S. aureus, CNS, P. acnes, or P. granulosum in acne vulgaris patients. In contrast, tests with other antibiotics revealed resistant strains with MICs surpassing 12.5 Ìg/ml.
It is worth noting that antibiotic resistance is a growing concern, particularly in the context of SSTIs. The emergence of multidrug-resistant strains poses significant challenges in the effective treatment of these infections. Therefore, periodic surveillance of antibiotic susceptibility patterns is crucial for guiding empirical therapy and ensuring the selection of appropriate antibiotics.24, 25
It is important to consider several limitations of the study. In vitro studies have inherent limitations in replicating the complexities of the human body, thus the results may not accurately reflect the clinical response. The study focused on specific infections and may not apply to other types or populations. Genetic factors and resistance mechanisms were not analyzed, limiting insights into treatment strategies. Pharmacokinetic and pharmacodynamic properties were not considered, which can impact clinical effectiveness. The sample size was relatively small, warranting caution in generalizing the results.
In summary, the study employed a comparative approach to assess multiple antibiotics used for SSTIs, providing comprehensive insights for antibiotic selection. It evaluated both ZOI and MIC, enhancing the understanding of antibiotic efficacy. The focus on relevant bacterial strains and prospective design strengthens the applicability and reliability of the findings. The study's results can guide future research and evidence-based treatment guidelines. However, further research considering larger sample sizes and additional factors is needed to optimize treatment strategies for these infections.
Conclusion
In conclusion, this prospective, comparative, in vitro study evaluated the antibiotic susceptibility patterns of common bacterial strains causing SSTIs. The findings demonstrated varying degrees of susceptibility to the tested antibiotics, including Nadifloxacin, Mupirocin, Clindamycin, and Fusidic acid. These results provide valuable insights into the selection of empirical treatment options for such infections. Nadifloxacin’s superior efficacy in the study can be attributed to its specific mode of action, broad spectrum of activity, excellent tissue penetration, favorable pharmacokinetic profile, and low risk of resistance development. These scientific properties make Nadifloxacin a valuable choice in the treatment of bacterial infections, particularly those involving the skin and soft tissues. However, it is essential to consider the limitations of in vitro studies and the need for further research to better understand antibiotic efficacy and resistance mechanisms in clinical settings. Overall, this study contributes to the knowledge base and can guide clinicians in making informed decisions regarding antibiotic therapy for SSTIs.
Conflicts of interest
Dr Rashmi is technical expert at Agilus Diagnostics Limited. All other authors are employees of Dr. Reddy’s Laboratories.
