Iranian Journal of Medical Microbiology

Staphylococcus aureus, in particular, methicillin resistant S. aureus (MRSA), is one of the main causes of hospital infections and community-acquired infections, with high morbidity and mortality rates worldwide (1). S. aureus infections cause difficult conditions to care and treat ulcers, damaged skin and soft tissue (2). MRSA are the principle cause of colonization and infection in the acute and chronic ulcerous lesions (4). Efflux Pump is one of the mechanisms of these bacteria in order to show antibiotic resistance (5). Many studies have investigated the inhibitory effect of some plant essential oils on efflux pumps in S. aureus strains (8-10). The simple way to evaluate the phenotypic effect of the efflux pump is the use of EtBr-Agar, which takes advantage agar plates and contains EtBr-increasing concentrations, which is known as the EtBr-Agar or cartwheel method (11, 12). Inside the plants, some compounds act in association with the antimicrobial agents, as they are responsible for disrupting the function of the efflux pumps in plant pathogens (7). Some plants have been involved in the treatment of skin diseases, and their effects have been proven like Matricaria chamomilla belongs to Asteracae family. This plant has antimicrobial and anti-inflammatory effects (13,14). The aim of this study was to investigate the effect of M. chamomilla alcoholic extract on phenotypes of MRSA strains isolated from skin lesions.

 

This study was performed on 30 samples of S. aureus collected from patients with skin lesions who referred to Shariati hospital in Tehran in the spring of 2017. The samples were characterized by different biochemical tests, including Gram stain, catalase, coagulase, DNase and mannitol fermentation, which led to identification and isolation. The antibiotics resistance of strains was assessed by disk diffusion method. To detect the phenotypic resistance of methicillin-resistant S. aureus, the susceptibility of the bacterial specimen to Cefoxitin disc (30 μg) in the Muller Hinton Culture medium was investigated. The M. chamomilla capitols were collected from Ardebil city and after taxonomic identification, they dried at 25°C in the shade and then powdered by the mechanical mill. The powder obtained from chamomilla capitols was mixed with 80% ethanol and then placed in a Soxhlet extractor. M. chamomilla alcoholic extract was prepared and MIC of it was obtained against MRSA strains by broth microdilution method. In order to investigate the activity of the efflux pumps phenotypically, all stains were evaluated by ethidium bromide containing agar using Cartwheel method (16). After identification of the strains with an activated efflux pump, the effect of M. chamomilla extract on the inhibitory activity of the pump was investigated. For this purpose, the dilutions 1/2 and 1/4 of the MIC of the extract were applied (according to the MIC level of each strain).

 

According to the results, the highest antibiotic resistance of S. aureus strains was respectively related to penicillin (83.3%), cefoxitin (33.3%), while the lowest resistance belongs to chloramphenicol which reported (3.3 %). among 30 S. aureus strains, 10 strains (33.3%) were MRSA (Fig. 1). The results of this study and the comparison with other reports on the prevalence of methicillin-resistant strains indicate increased methicillin-resistant strains, one of the reasons of which has been due to the excessive use of antibiotics in recent years. MIC of M. chamomilla extract was tested for the studied strains and result obtained between 128-64 μg/ml. The antimicrobial effect of M. chamomilla extract and essential oil against a range of bacteria and fungi has been investigated and confirmed (20, 21).
In this study, ethidium bromide cartwheel assay was performed to detect the phenotypic activity of the efflux pump in S. aureus strains. Based on the results of this study, three strains were highly active among 30 strains, and did not exhibit fluorescence in all dilutions of ethidium bromide, which means they had strong efflux pumps. Among these three strains, one was MRSA. With the effect of 1/2 MIC of M. chamomilla extract on the strains with a strong efflux pumps, the two stains were reported as non-actives. M. chamomilla extract on the MRSA strain efflux pump was ineffective. (Figs. 2 and 3). Evaluation of 1/4 MIC of M. chamomilla extract had no effect on efflux pump activity. Khan et al. (2006) showed that the piperine, a plant alkaloid of the Piperaceae family, at a concentration of 25 mg/L reduced two-fold the MIC of S. aureus compared to the ciprofloxacin antibiotic. This decrease in MIC is due to the increase in the antibiotic concentration of ciprofloxacin in the bacterium, because of the inhibitory effect of the efflux pumps (28). Smith et al. used the Totarol isolated from the immature cones of Chamaecyparis nootkatensis to inhibit the NorA S. aureus pump. They used MIC analysis of ethidium bromide in combination with Totarol to evaluate pump inhibition and reported that the compound studied had antimicrobial activity and also inhibited the NorA pump (26).

Figure 1. Antibiotic resistance patterns of Staphylococcus aureus strains


Figure 2. Fluorescent plates containing concentrations of 0.25 to 2.5 g/L of ethidium bromide and microbial suspension of Staphylococcus aureus strains: strains 13, 14 and 16 did not show fluorescent activity in all dilutions of ethidium bromide and became known as strong strains. Each growth line belongs to one isolate. Transparent culture lines indicate inactivation of the effluent pump and opaque cultivation lines indicate that the effluent pump system is active. Strain 14 was MRSA.



Figure 3. Fluorescent plate images containing ethidium bromide and microbial suspension with MIC concentration of 1/2 M. chamomilla extract of each strain: strains 13 and 16 showed fluorescent activity in all dilutions of ethidium bromide and were identified as inactive. MRSA strain 14 remained active.
 

There are major challenges to find new inhibitors. Considering the antimicrobial effects of plant compounds on inhibiting efflux pump, bacteria that were initially resistant to antibiotics can be sensitive later and, if this approach is successful in the future, it could be considered as an important alternative in the treatment of some infections caused by drug-resistant strains. It is also important to determine the expression level of these genes encoding efflux pumps among phenotypically active isolates, and that the role of synergy between efflux pumps and other antibiotic resistance mechanisms, to achieve this high resistance level, should not be ignored. Further studies are suggested to investigate the genotypic expression of efflux pumps in methicillin-resistant S. aureus strains.

 

The authors would like the staff of Microbiology Research Laboratory of Azad Islamic University, Varamin branch and also Mr. Omid Hosseini, the technician of the Research Laboratory of Shahid Beheshty University. The ethical code for this research was IR.IAU.VARAMIN.REC.1396.3.

 

The authors reported no conflict of interest.