Isolation and Characterization of Probionibacterium acne from Acne Vulgaris Patients

In this study, 110 skin swabs were obtained from patients suffering from acne vulgaris from consultation unit in Merjan teaching Hospital, and allergy specialized center in Al-Hilla from both sexes . It was found that acne vulgaris was higher in female (62.7%) and more prevalence in (16-20) years patients and lower in older especially after 30 years age where there is only one case. Three isolates of anaerobic gram positive Probionibacterium acnes were identified and diagnosis by culture and biochemical tests, all bacterial strains possessed capsule and produce lipase and protease enzymes and contained the thired colonization factor antigen (CFA-III) while only one strain produce a narrow zone of β-hemolysis . The effect of some antibiotics on Probionibacterium acnes was investigated, and the results showed that all isolates were sensitive to Tetracycline ,Deoxcycline, Chloromphenecol, Neomycine, and Kephalexin, while they were resistant to Erythromycin, Fusidic acid, Vencomycin, Lincomycin and Gentamycin. Also retinoic acid effect on bacterial growth was studied. It was stated that lactic at high concentration 20μg/ml could cause inhibition of Probionibacterium acnes growth. Also temperature over 50 C° would kill the bacteria. Introduction : Acne is a chronic inflammatory disease of the pilosebaceous unite, primarily of the face, nose, upper back, chest, shoulders and upper arms. It is one of the commonest dermatoses affecting the teenage population (Sehgar & Jain, 1995). On the face it occurs most frequently on the cheeks, and in lesser degree on the nose, forehead and chin (Andrews, 1990). Acne starting usually between the ages of 12 and 14 years, tending to be earlier in females. The peak age for severity in females in 16-17 and in males 17-19 years (Lucky, et al.,1991). Acne frequently occurs in individuals with high sebum production and bacteria is observed at acne regions. Products generated by ingestion and degradation of sebum by Propionibacterium acnes are considered to cause inflammation around pores, and aggravate the symptoms (Ingham, et al.,1992). Endocrine, keratinization, and bacterial factors are considered to be the major factors in acne development. An increase in testosterone after puberty is considered to increase sebum productivity of sebaceous gland . Free fatty acid produced by P.acnes derived lipase, induced impairment of keratinization and sebum accumulated in the hair follicular duct (Downing, et al.,1986). When untreated, acne usually lasts for several years until it spontaneously remits Adolescent acne (acne vulgaris) has a strong tendency to be hereditary and is less likely to be seen in Asians and dark skinned people . The pliosebaceous follicle is the target organ in acne, explaining the distribution of acne primarily on the face, chest and areas with the greatest concentration of pliosebaceous glands . Patients and Methods: One hundred and ten samples were collected from acne patients who attended the consultation unit of Merjan teaching Hospital in Babylon province during the period of November 2004 to April 2005; their ages ranged from 13-30 years old. The patients were suffering from sever-moderate inflammatory acne. After an incubation period ,rod shaped bacteria was stained with Gram stain then with Albert stain, then tested with biochemical test including ; Catalase, Indole production, Nitrate reduction, Esculin hydrolysis, then to detect some virulence factors and adhesions such as capsule, by negative stain (Cruickshank, et al., 1975), haemoagglutination test, production of hemolysin, sidrophore production and colonization factor antigens. The bacterial isolates were also tested for production of some enzymes especially lipase and protease, also the effect of Retinoic acid and many other antibitics were used to test the sensitivity of the bacteria (Maccfeiden et al., 1995). 1.Sidrophore production assay : M9 agar was used for assay wich is supplemented with 0.25 gm glucose after sterilization, 200μm of dipyyridyl is added ( Nassif et al.,1989) . 2. Retinoic acid assay : A. Nutrient broth is prepared and distributed in tubes and the retinoic acid is added to each tube at various volume to gain the final concentration ( 10, 20, 40, 80 and 100 mg/ml). B. Positive control is prepared by using Nutrient broth from Retinoic acid. C. The tubes in item 1 and 2 are inoculated with 0.5 ml of bacterial suspension and then incubated anaerobicaly for 24 hour at 37C°. D. After incubation, the absorbency was red at wave length 520 nm. E. Draw standard curve. 3.Assay for colonization factor antigens : Assay for colonization factors was used depending on (Smyth, 1982) as follow : A.CFA/I : I.Inoculate bacterial strain on tryptic soy agar which is incubated in 37C° for 24 h. II.RBCs suspension was prepared from group A with phosphate buffer saline (repeated three times) the 3% (v/v) of RBCs was prepared. III. Bacterial suspention from that grown on TSA , and mixed with 0.1M NaCL to determine RBCs agglutination test and was castigated on colonies factor antigen type one. IV.On clean slide mixed one drop of bacterial suspension with one drop of RBCs suspension and one drop of 0.15M NaCL( without D-manose). V. Examine the agglutination at room temperature and from 1/2 –2 min. B. CFA/II : To determine this factor the same steps were followed except , chicken blood was used instead of group A blood. C.CFA/III : Also the same steps were used except that after growing on TSA mix with 0.1 M of NaCL. And using the tannic acid to visualize the agglutination of human RBCs type A blood group. Results and Discussion : Acne is a common inflammatory skin disease due to inflammation of blocked hair follicles. Each hair follicle is associated with a sebaceous gland which secretes a conditioning oil (sebum) into the follicle. The condition usually starts in adolescence in both male and female. In this study the distribution of acne vulgaris was higher among female than male (62.7% and 37.2% ) respectively. This result was correlated with those results obtained by Kligman (1974) who had pointed that acne prevalence was more among female than in male, also Borton et.al., 1971 said that acne was developed earlier in females than in males. Furthermore, the distribution of this disease among patient ages was also studied. Most cases were in patients ages (16-20) years old in both sexes. Burton results were identical to our results obtained by this study about the severity of acne in the ages between 15-17 years in females, and 16-20 years in male. Acne problems appear in older patients in mid-twenties (20.9%), Rea et.al., 1997 said 18% had late-onset (more 20 years). Also in this study (4.5%) of patients had their acne between (20-35) . This result was close to Stern, 1992, result’s which have 5% and 1% of patients who had acne in the same age groups. All these results and others were listed in table ( 1 ). Table (1 ): Distribution of acne infection among age and sex.