Acne vulgaris, commonly known as acne, is one of the most common dermatological disorders affecting about 85% of adolescents worldwide 1. It has significant psychological implications ranging from anxiety and depression up to suicidal attempts 2. The pathogenesis of acne vulgaris is complex and multifactorial. Excess sebum production, follicular epidermal hyperproliferation, inflammation, and bacterial colonization are the most accepted explanations 3. Although acne is not an infectious disease, three major organisms have been isolated from the pilosebaceous ducts of acne patients – Propionibacterium acnes, Staphylococcus epidermidis, and Malassezia furfur4. Staphylococcus aureus is also one of the microorganisms commonly isolated from acne lesions 1. One reason for the lively debate about their role in the pathogenesis of acne is the fact that all these microorganisms are present in normal skin 5–7. However, many studies have confirmed the pivotal role of bacteria in the initiation and maintenance of acne lesions 8–11. Besides, in-vitro evidences suggest a pathogenic role of staphylococci in the development of acne 12,13.
Evidence supporting staphylococcal involvement is still controversial, but topical and systemic antibiotics have been extensively prescribed to treat acne. Eventually, this led to the appearance of resistant and even life-threatening strains of bacteria – for example, methicillin-resistant S. aureus14,15. To use appropriate medications against staphylococci, its virulence factors should be adequately determined. Documented virulence factors include the presence of extracellular enzymes such as lipase and protease as well as the ability to form biofilms 16.The enzyme lipase is responsible for the hydrolysis of lipid emulsions composed of long-chain fatty acids. It usually interacts with the skin environment rich in lipids and other polymers 17, releasing oleic acid and other free fatty acids into the skin surface that have pathogenic activity 8,18,19. This activity may contribute to resistance to antimicrobial agents 20. Lipase-encoding genes are suggested to be among the upregulated genes involved in biofilm formation in S. aureus21, and antilipase serum was found to inhibit the cascade of biofilm formation 22. Lipase produced by S. epidermidis was also found to be associated with colonization and biofilm development 23. By coating the host tissues, this biofilm promotes infection persistence and multidrug resistance 24.
Most of our knowledge about staphylococci is obtained from strains isolated from various sites such as blood, infected devices, conjunctiva, and skin 25–27. On the other hand, little is known about virulence factors of strains isolated from acne lesions and their possible contribution to the development of antimicrobial resistance. The aim of this study was to investigate lipase enzyme activity in staphylococcal isolates and assess the association between antibiotic resistance and lipase activity in S. aureus and S. epidermidis strains sampled from acne patients.
Patients and methods
This was a cross-sectional study that involved 102 clinically diagnosed acne patients who visited the outpatient Clinic of Dermatology at Beni-Suef University Hospital between June 2014 and September 2014. Exclusion criteria included patients younger than 16 years, patients with endocrine disorders, and those who did not stop their medications 3 months before the study. Patients were informed about the aim of the study in detail, and consent was obtained from all of them. The study was approved by the Ethics Committee of Beni-Suef University. First, patients were asked to complete a simple questionnaire including questions about their age, sex, duration of disease, and history of antibiotic treatment. The degree of acne severity was determined according to the global acne grading system 28.
Specimen collection and processing
Samples were collected from an inflammatory papule or pustule. After cleansing with 70% ethanol, lesions were punctured with a hypodermic needle, and the contents were collected using a comedone extractor. Samples were transferred immediately into a pouch containing Amies medium to the Microbiology Laboratory of Beni-Suef University Hospital.
Culture of specimens
Cultures were incubated under both aerobic and anaerobic conditions for isolation and identification of different organisms. Identification of cultures was based on colony morphology, Gram staining, and biochemical tests. For staphylococcal isolation, all samples were cultured on mannitol salt agar and blood agar (Oxoid Laboratories, Basingstoke, Hampshire, UK). These plates were incubated at 37°C in aerobic conditions for up to 48 h, and were examined for bacterial growth. Besides, strains were grown on anaerobic blood agar (CM0972; Oxoid Laboratories, UK) for isolation of P. acnes. Cultures were incubated at 37°C in an anaerobic cabinet (MACS MG 1000; Don Whitley Scientific, Don Whitley Scientific Limited, West Yorkshire, UK), in an atmosphere of 80% N2, 10% CO2, and 10% H2. Isolates were examined using API 50 CHO biochemical identification systems (Biomérieux, Boston, Cambridge, USA) in accordance with the manufacturer’s instructions. MacConckey agar was used for Gram-negative isolates.
Identification of bacterial isolates
Identification of Gram-positive staphylococcal strains was carried out by conventional microbiological standard tests – for example, colony morphology analysis, Gram staining, and biochemical tests for catalase and coagulase. Each isolated strain was stored in two ways: inoculation on a slope of nutrient agar stored in the refrigerator and subcultured frequently until used for culture and sensitivity tests, and sterile glycerol broth suspension stored at 70°C for screening lipolytic effect, lipase production, and activity. S. aureus and S. epidermidis strains were detected as described previously 29. Both S. aureus and S. epidermidis strains were catalase positive. S. aureus strains were coagulase positive, whereas S. epidermidis strains were coagulase negative.
Antimicrobial susceptibility test
For the antimicrobial susceptibility test, McFarland 0.5 turbidity standard bottles, antibiotics discs for antibiotic susceptibility testing (Oxoid Laboratories, UK), and Mueller–Hinton agar (Oxoid Laboratories, UK) were used. S. aureus and S. epidermidis isolates were subjected to a disk-diffusion susceptibility test with the following antibiotics: azithromycin, clarithromycin, tetracycline, doxycycline, minocycline, trimethoprim sulfamethoxazole, cefotaxime, clindamycin, penicillin, streptomycin, chloramphenicol, linezolid, ciprofloxacin, and norfloxacin (Oxoid Laboratories, UK). Inhibition halos were interpreted according to the Clinical Laboratory and Standards Institute guidelines, 2014 http://clsi.org/wpcontent/uploads/sites/14/2014/12/CLSI_Fall_2014_Catalog_Web1.pdf. P. acnes culture, identification, and isolation conditions have been discussed in a previous study 30.
Screening of lipolytic bacteria
Subculturing of samples was carried out in nutrient broth media. The pH of the medium was adjusted to 7 with 0.1 mol/l NaOH. The isolation process was performed by serial dilution of samples on tributyrin agar plates. The composition of tributyrin agar medium is (per liter) 5 g peptone, 3 g yeast extract, 10 ml tributyrin, and 15 g agar. Culture plates were incubated at 30°C. Colonies showing clear zones around them were selected, purified on tributyrin agar plates, and transferred to agar slants. Isolates having a clearing zone were cultured in liquid media, and the level of lipase production was determined from the cell-free culture supernatant fluid as described earlier 31.
The composition of the production medium used in this study was as follows: (w/v%) pepton, 0.2; NH4H2PO4, 0.1; NaCl, 0.25; MgSO4·7H2O, 0.04; CaCl2·2H2O, 0.04; olive oil, 2.0 (v/v), pH 7.0, and one to two drops Tween 80 as an emulsifier. Overnight cultures were suspended in 5 ml of sterile deionized water and used as inoculum for precultures to obtain an initial cell density to adjust the turbidity of 0.5 McFarland standards. Submerged microbial cultures were incubated in 500-ml Erlenmeyer flasks containing 100 ml of liquid medium on a rotary shaker (150 rpm) and incubated at 30°C. After 24 h of incubation, the culture was centrifuged at 10 000 rpm for 20 min at 4°C, and the cell-free culture supernatant fluid was used as the source of extracellular enzymes.
Assay of lipase activity
Lipase activity was determined spectrophotometrically at 30°C using p-nitrophenyl palmitate (pNPP) as substrate. The reaction mixture was composed of 700-μl pNPP solution and 300 μl of lipase solution. The pNPP solution was prepared by adding solution A (0.001 g pNPP in 1 ml isopropanal) to solution B (0.01 g gum arabic, 0.02 g sodium deoxycholate, 50 μl Triton X-100, and 9 ml of 50 mM Tris-HCl buffer, pH 8) with stirring until completely dissolved. Next, absorbance was measured at 410 nm for the first 2 min of reaction. One unit was defined as that amount of enzyme required to liberate 1 μmol of pNPP per minute ([Latin Small Letter Open E]: 1500 mol/l/ cm) under the test conditions as previously described 29.
Data were analyzed using statistical package for the social sciences (SPSS, version 19). Frequency distribution with its percentage and descriptive statistics with mean and SD were calculated. χ2-test and t-test were performed whenever needed. P values of less than 0.05 were considered significant.
A total of 102 acne patients, attending the dermatology clinic of Beni-Suef University Hospital, from June 2014 to September 2014, were recruited for the study. Eighty (78.4%) participants were female and 22 (21.6%) were male. Patients’ age ranged from 12 to 33 years with a mean of 20.4±5.61 years. The disease duration varied from 1 to 132 months, with a mean duration of 34.19±11.26 months.
Of our 102 patients, 28 (27.5%) patients were living in urban districts and 74 (72.5%) were living in rural areas. Almost 81.3% of patients were literate and only 18.7% were illiterate, and nearly 78.5% of the patients were not working. Ninety-three (91.2%) patients were diagnosed clinically as having moderate acne, whereas nine (8.9%) patients had severe acne. The number of patients who reported as being treated with topical antibiotics was 68 (66.7%), whereas 22 (21.6%) patients received combined topical and systemic treatment. Only 44 (43.1%) patients reported of having a positive family history of acne.
Of the 102 specimens isolated, 28 (27.5%) were positive for P. acnes, 18 (17.6%) were positive for S. aureus, and nine (8.8%) lesions showed the presence of S. epidermidis. Other isolates included Spirochetes spp., micrococci, and Klebsiella pneumoniae in five (4.9%) cultures, whereas Citrobacter spp., streptococci, and Diphtheroid spp. were detected in four (3.9%) cultures. Klebsiella oxytoca was isolated once, whereas 19 (18.6%) isolates showed no growth. Three specimens showed P. acnes along with S. epidermidis, and two specimens showed P. acnes along with S. aureus.
Data on sociodemographic characteristics, family history, diseases duration, and previous acne treatment of patients with S. aureus and S. epidermidis are illustrated in Table 1.
Lipase enzyme was detected in nine (100%) isolates of S. epidermidis, and 15 out of 18 (83.3%) isolates of S. aureus were lipase positive and only three (16.7%) were lipase negative.Of the nine lipase-positive S. epidermidis specimens, six (66.7%) were lipase active and three (33.3%) were lipase inactive, and out of the 15 lipase-positive S. aureus specimens 10 (66.7%) were lipase active and five (33.3%) were lipase inactive.
Our results also pointed out that acne lesions with S. epidermidis showed high sensitivity to azithromycin and clarithromycin in six (66.7%) cases, followed by minocycline and cefotaxime in five (55.6%) cases, whereas both tetracycline and clindamycin were the least sensitive with one (11.1%) specimen for each. On studying the association between lipase activity and susceptibility of S. epidermidis to different antimicrobials, apart from lipase activity in all resistant specimens to trimethoprim sulfamethoxazole compared with its activity in only one sensitive specimen to the same antimicrobial (P<0.05), activity of lipase enzyme was shown not to be related to the susceptibility of S. epidermidis-positive isolates to the other antimicrobials (P>0.05) (Table 2).
In addition, acne lesions with S. aureus showed the highest sensitivity to penicillin in 11 (61.1%) cases, to linezolid and clarithromycin in nine (50%) cases each, and tetracycline in eight (44.4%) cases, whereas no specimens were sensitive to minocycline. On the other hand, activity of lipase enzyme was statistically not related to the susceptibility of S. aureus-positive isolates to any of the studied antimicrobials (P>0.05) (Table 3).
Of the 18 isolates with S. aureus, cross-resistance was detected in eight isolates between erythromycin and azithromycin, nine isolates between erythromycin and linezolid, and 10 isolates between erythromycin and cefotaxime. In addition, eight isolates showed cross-resistance between tetracyclin and clindamycin, whereas nine isolates showed cross-resistance between tetracyclin and minocycline. Doxycycline showed cross-resistance with azithromycin in nine isolates, with norfloxacin in 11 isolates, with cefotaxime in 12 isolates, and with minocycline in 14 isolates.
Sociodemographics, family history, and history of treatment were not related to either presence of lipase or lipase activity in both S. epidermidis and S. aureus (P>0.05).
In a previous study, we demonstrated that bacteria other than P. acnes constituted more than half of the isolated bacteria from patients with acne 30, which is consistent with the previous literature 1,32,33 and is also contradicted by a few studies 34–36. For instance, Moon et al.32 found that, of 100 bacterial isolates from Korean patients, S. epidermidis was the most common – from 36 patients, S. aureus was isolated in eight cases. On the other hand, Toyne et al.34 who studied Australian adolescents and Mendoza et al.36 who studied Colombian patients found much lower rates of species other than P. acnes. Such variations in results emerging from studying different populations do not only implicate the polymicrobial nature of acne lesions, but also highlight the impact of geographical distribution of the bacteria involved in acne vulgaris.
Although S. epidermidis was previously considered a nonvirulent, cutaneous commensal, many of its opportunistic strains have been linked to serious infections associated with synthetic medical devices, including prosthetic heart valves and intravenous catheters 37. Moreover, it is one of the organisms commonly isolated from acne lesions 38,39. On the other hand, S. aureus is the most common cause of hospital-associated infections 40. In addition to causing life-threatening morbidities 41, S. aureus can rapidly develop resistant strategies 42. Fanelli et al.12 demonstrated that 43% of acne patients were colonized with S. aureus, and the microorganism was isolated from acne lesions in several studies 32,38.
Staphylococcal strains produce a wide range of extracellular enzymes, including lipase, which is considered an important virulence factor. Staphylococcal lipase can interfere with phagocytosis of human granulocytes 43. It is also responsible for colonization and persistence of bacteria on the skin 44 as well as dermal and epidermal tissue invasion 45 in addition to producing irritant fatty acids 46. It is also linked to biofilm formation, which is considered the major pathogenic factor of staphylococci. This biofilm not only facilitates the adherence of these microorganisms, but also protects them from the host’s immune system and antimicrobial therapy 21–24.
In the present study, lipase was detected in all isolates of S. epidermidis and in 83.3% of isolated S. aureus strains – the enzyme activity was higher in S. epidermidis isolates than in S. aureus strains. Our findings are in agreement with Saising et al.47 who concluded that coagulase-positive staphylococci released lipase enzyme more often than coagulase-negative strains in acne patients. The authors found that strains isolated from the face secreted the enzyme at a higher rate than staphylococcal strains isolated from other parts of the body 47. This may shed more light on the role of lipase in initiating or aggravating acne lesions.
Besides, our study demonstrated variable patterns of antimicrobial susceptibility among the isolated bacteria. S. epidermidis showed high sensitivity to azithromycin and clarithromycin, whereas most strains were resistant to antibiotics commonly prescribed in acne treatment: tetracycline, doxycycline, and erythromycin with resistance of 88.9, 66.7, and 66.7%, respectively. Moon et al.32 concluded that S. epidermidis strains were mostly resistant to erythromycin, tetracyclin, and doxycycline with rates of 58.3, 30.6, and 27.3%, respectively. Nakase et al.39 found that S. epidermidis was highly resistant to erythromycin and clarithromycin (58.6%). A study from Brazil on S. epidermidis in ICUs showed that 82.6% were resistant to gentamicin, 79.6% to erythromycin, 74.5% to clindamycin, and 71.4% to ciprofloxacin, whereas all the isolates were susceptible to vancomycin 48.
On the other hand, our findings suggest that S. aureus isolates were highly sensitive to penicillin, linezolid, and clarithromycin, but highly resistant to minocycline, erythromycin, and doxycycline. Moon et al.32 found that S. aureus from acne lesions showed the highest resistance to tetracyclin, doxycycline, and erythromycin with values of 87.5, 87.5, and 75%, respectively. In a study from Iran, S. aureus from acne lesions were mostly resistant to doxycycine and tetracyclin with values of 72.2 and 69.4%, respectively 13. S. aureus isolated from other body sites of acne patients showed relatively different patterns of resistance. Fanelli et al. 12 who isolated S. aureus from the throat and nose of acne patients in USA showed that nasal isolates showed higher rates of resistance to antibiotics, particularly to clindamycin and erythromycin.
A study by Michelim et al. 48 on 98 ICU isolates found that 90% of the S. epidermidis strains were lipase positive, but no significant association was demonstrated between lipase activity and bacterial drug resistance. Our study also detected relatively higher rates of lipase positivity and activity in resistant strains; however, our limited sample size prevented us from achieving a statistically significant association between lipase enzyme and staphylococcal response to various antimicrobial agents.
In conclusion, our study confirmed the findings of previous studies on the pathogenicity of staphylococcal strains in acne. We also detected high resistance rates of staphylococcal strains to different antimicrobial agents. Although no statistically considerable association was detected between lipase enzyme and development of antimicrobial resistance, future studies should include larger sample sizes and isolates from different body areas.
Conflicts of interest
There are no conflicts of interest.
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