# Molecular and Immunological Characterization ofStaphylococcus aureus in Pediatric Atopic Dermatitis: Implications for Prophylaxis and Clinical Management

Authors: Chiara Pascolini; JoLinda Sinagra; Simone Pecetta; Valentina Bordignon; Alessandra De Santis; Laura Cilli; Viviana Cafiso; Grazia Prignano; Bruno Capitanio; Claudio Passariello; Stefania Stefani; Paola Cordiali-Fei; Fabrizio Ensoli
Journal: Clinical and Developmental Immunology
 (2011)

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## Abstract

S. aureus represents a critical cofactor in atopic dermatitis (AD). In this paper, the prevalence of S. aureus infection/colonization was evaluated in 117 children as well as in their cohabitants, in order to assess the value of S. aureus characterization in predicting disease onset and severity and in  providing indications for prophylaxis. Results showed that children with AD as well as their cohabitants had a significantly greater incidence of S. aureus infection/colonization as compared to controls. The genetic characterization showed a virtual identity of the bacteria strains collected at different sites of the patients with those found in the cohabitants, suggesting both a direct transmission between the nasal reservoir and the lesions in the same atopic subject and a risk for reinfection within family cohabitants. These data stress the need of preliminary laboratory assessment and posttherapy control in both AD patients and their close contacts for effective S. aureus eradication.


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## Body

## 1. Introduction

Atopic dermatitis (AD) is an inflammatory disease affecting the skin and characterized by impaired epidermal barrier function and cutaneous inflammation [1, 2]. It is clinically characterized by an early onset, mostly occurring before 5 years of age [3]. The prevalence of AD has increased in industrialized countries during the past three decades, and among Italian schoolchildren the estimated prevalence is presently of about 5.8% [4]. The eczematous skin of the patients is highly susceptible to bacterial colonization, and Staphylococcus aureus (S. aureus) represents the most frequent isolate [5–8]. S. aureus is a Gram-positive opportunistic bacterium that, although part of the normal flora of the skin, can give rise, as a consequence of clonal evolution, to virulent strains characterized by the expression of virulence factors and the acquisition of resistance to a number of different drugs including β-lactams [9]. S. aureus infection appears to play an important role in the pathogenesis of AD by either causing or exacerbating skin inflammation [10–13].  In fact, S. aureus toxins have potent superantigenic properties (e.g., staphylococcal enterotoxin SEA, SEB, SEC, SED, and toxic shock syndrome toxin (TSST-1)) and may induce monocytes and lymphocytes activation, which in turn are induced to produce several inflammatory cytokines [14, 15]. In a healthy skin, this process causes an inflammatory reaction with a clinical outcome similar to that seen in AD [10].Although the impact ofS. aureus infection in the clinical manifestation of AD is still debated, antibiotic-corticosteroid combination therapy against S. aureus represents an important component of the therapeutic approach to AD [16] and appears effective against both the occurrence of secondary infections and the severity of the disease [17]. However, it should be also considered that the clinical benefits appear often transient and that the onset of an antibiotic resistance represents a serious emerging problem in long-term therapy [18, 19] suggesting the need of an accurate microbiological analysis for a most adequate and effective therapy.In this study, we investigated the prevalence and relationship betweenS. aureusinfection/colonization, in both skin lesions and anterior nares, and the clinical severity of the disease in children with AD as compared to a control group of asymptomatic children and examined the influence of S.  aureuscolonization in the family members as a potential source for reinfection. The results indicated that children with AD had a significantly greater prevalence of S. aureus infection/colonization as compared to controls. The genetic characterization confirmed the identity of bacteria strains collected from the patients with those found in the cohabitants, suggesting both a direct transmission between the lesions in the same atopic subject and a risk for reinfection within family cohabitants. These results emphasize the importance of a preliminary laboratory assessment and posttherapy control in both AD patients and their close contacts for effective S. aureus eradication.


## 2. Patients and Methods

### 2.1. Patients and Controls

Patients incoming the Pediatric Outpatient Clinic at the San Gallicano Dermatology Institute were examined during a two-year period (2007-2008). One hundred and seventeen children aged between 3 months and 12 years (42 patients were under 12 months of age) of both sexes, suffering from mild to severe AD, were included in the study. Diagnosis of AD was based on the criteria described by Hanifin and Rajka [20]. The severity of AD was measured by the scoring atopic dermatitis index (SCORAD) of the European Task Force on atopic dermatitis. Briefly, a numeric score (1–10) was assigned to (a) extent  and (b) intensity  of skin lesions and (c) to subjective symptoms like pruritus and sleep loss. The final SCORAD was calculated by the following equation: a/5 + 7b/2 + c. In the present study, the clinical expression of disease was classified as low, medium, or high according to SCORAD values ranging between 0 and 15, 15, and 40 or >40, respectively. Patients did not receive any steroid or antibiotic therapy in the last two months before the initiation of the present study. Parents and cohabitant family members of AD children were asked to voluntarily participate to the study by allowing skin and nose sampling and examination aimed at S. aureus isolation and characterization. One hundred and ten subjects of 37 families accepted to enter the study. S. aureus strains were also examined in asymptomatic carriers consisting of 90 healthy children, of both sexes (39/51 M/F), aged between 3 months and 12 years and 80 adults, of both sexes, aged between 25 and 50, attending the Outpatient Dermatology Clinic to control pigmented lesions. All samples were obtained by sterile swabs, from skin and nose. All patients parents and healthy volunteers gave their written informed consent before study initiation.


### 2.2. Microbiological Analysis

Swabs were collected from lesions, normal skin areas as well as from nares of atopic children. Nasal swabs were performed in their cohabitants or control subjects. Samples were plated on enriched (blood agar), selective (Mannitol Salt Agar) and differential (MRSA ChromID-BioMérieux, France) media and incubated for 24–48 h at 37°C. Identification and antimicrobial susceptibility ofS. aureus were performed by an automated diagnostic system (Card AST P580, Vitek 2, BioMérieux, France) and included the most frequently used drugs for therapeutic use against S. aureus. Bacteria isolates were classified as Methicillin-resistant Staphylococcus aureus (MRSA) on the basis of resistance to oxacillin (≥4 μg/mL) and positivity of the Penicillin-Binding Protein latex agglutination test (PBP2’, Oxoid, UK).


### 2.3. Molecular Characterization

Molecular analysis was performed through Multiplex-PCR as previously described [21] in order to assess the expression of the following genes: capsular antigen (cap 5-cap 8); agr-group (agr); adhesins (hls-spa-ica A-atl-cna-sdr E-sdr C-fnb A-clf A/B); toxins (eta-sea-sej-sec-sed-sek-seq-tst-splB-lukE); Panton-Valentine leukocydin-PVL (lukS/F). Biofilm production was assessed as previously described [22–24].


### 2.4. Immunoassay

To detect the production of staphylococcal enterotoxins AD and toxic shock syndrome toxin-1 (TSST-1) isolates were incubated in tryptone soya broth and brain heart infusion, respectively, for 24 h at 37°C. Supernatants were tested for the presence of exotoxins by two specific reverse passive latex agglutination commercial kits (SET-RPLA and TST-RPLA) according to the instructions of the manufacturer (Oxoid, UK). The sensitivity of these agglutination assays is 0.5 ng/mL (SET-RPLA) and 2 ng/mL (TST-RPLA), respectively.


### 2.5. PFGE Analysis

Genotyping of isolatedS. aureusstrains was performed by pulsed-field gel electrophoresis (PFGE) after DNA digestion with SmaI (Fermentas ER0661), following the CDC (Center of Disease Control and Prevention, USA) protocol [25]. Briefly, electrophoresis was performed with pulses ranging from 5 to 45 seconds at a voltage of 6V/cm at 14°C for 23 hours using the PFGE instrument CHEF DR II (Bio-Rad, USA). The gels were stained for 20 minutes with ethidium bromide solution and observed under UV illumination.


## 2.1. Patients and Controls

Patients incoming the Pediatric Outpatient Clinic at the San Gallicano Dermatology Institute were examined during a two-year period (2007-2008). One hundred and seventeen children aged between 3 months and 12 years (42 patients were under 12 months of age) of both sexes, suffering from mild to severe AD, were included in the study. Diagnosis of AD was based on the criteria described by Hanifin and Rajka [20]. The severity of AD was measured by the scoring atopic dermatitis index (SCORAD) of the European Task Force on atopic dermatitis. Briefly, a numeric score (1–10) was assigned to (a) extent  and (b) intensity  of skin lesions and (c) to subjective symptoms like pruritus and sleep loss. The final SCORAD was calculated by the following equation: a/5 + 7b/2 + c. In the present study, the clinical expression of disease was classified as low, medium, or high according to SCORAD values ranging between 0 and 15, 15, and 40 or >40, respectively. Patients did not receive any steroid or antibiotic therapy in the last two months before the initiation of the present study. Parents and cohabitant family members of AD children were asked to voluntarily participate to the study by allowing skin and nose sampling and examination aimed at S. aureus isolation and characterization. One hundred and ten subjects of 37 families accepted to enter the study. S. aureus strains were also examined in asymptomatic carriers consisting of 90 healthy children, of both sexes (39/51 M/F), aged between 3 months and 12 years and 80 adults, of both sexes, aged between 25 and 50, attending the Outpatient Dermatology Clinic to control pigmented lesions. All samples were obtained by sterile swabs, from skin and nose. All patients parents and healthy volunteers gave their written informed consent before study initiation.


## 2.2. Microbiological Analysis

Swabs were collected from lesions, normal skin areas as well as from nares of atopic children. Nasal swabs were performed in their cohabitants or control subjects. Samples were plated on enriched (blood agar), selective (Mannitol Salt Agar) and differential (MRSA ChromID-BioMérieux, France) media and incubated for 24–48 h at 37°C. Identification and antimicrobial susceptibility ofS. aureus were performed by an automated diagnostic system (Card AST P580, Vitek 2, BioMérieux, France) and included the most frequently used drugs for therapeutic use against S. aureus. Bacteria isolates were classified as Methicillin-resistant Staphylococcus aureus (MRSA) on the basis of resistance to oxacillin (≥4 μg/mL) and positivity of the Penicillin-Binding Protein latex agglutination test (PBP2’, Oxoid, UK).


## 2.3. Molecular Characterization

Molecular analysis was performed through Multiplex-PCR as previously described [21] in order to assess the expression of the following genes: capsular antigen (cap 5-cap 8); agr-group (agr); adhesins (hls-spa-ica A-atl-cna-sdr E-sdr C-fnb A-clf A/B); toxins (eta-sea-sej-sec-sed-sek-seq-tst-splB-lukE); Panton-Valentine leukocydin-PVL (lukS/F). Biofilm production was assessed as previously described [22–24].


## 2.4. Immunoassay

To detect the production of staphylococcal enterotoxins AD and toxic shock syndrome toxin-1 (TSST-1) isolates were incubated in tryptone soya broth and brain heart infusion, respectively, for 24 h at 37°C. Supernatants were tested for the presence of exotoxins by two specific reverse passive latex agglutination commercial kits (SET-RPLA and TST-RPLA) according to the instructions of the manufacturer (Oxoid, UK). The sensitivity of these agglutination assays is 0.5 ng/mL (SET-RPLA) and 2 ng/mL (TST-RPLA), respectively.


## 2.5. PFGE Analysis

Genotyping of isolatedS. aureusstrains was performed by pulsed-field gel electrophoresis (PFGE) after DNA digestion with SmaI (Fermentas ER0661), following the CDC (Center of Disease Control and Prevention, USA) protocol [25]. Briefly, electrophoresis was performed with pulses ranging from 5 to 45 seconds at a voltage of 6V/cm at 14°C for 23 hours using the PFGE instrument CHEF DR II (Bio-Rad, USA). The gels were stained for 20 minutes with ethidium bromide solution and observed under UV illumination.


## 3. Statistical Analysis

The statistical treatment of the results gathered from the different groups of subjects as well as the different clinical outcomes was performed by applying contingency tables and Fisher’st test (between two groups) or χ2 test (among three groups). The analysis has been performed by the GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, Calif, USA).


## 4. Results

Of the 117 patients analyzed, 66 (57%) were found to harborS. aureus(Table 1). In particular, we had positive isolation from skin lesions and nasal swabs in 47 patients (40.1%), while 19 patients (16.2%) had only nasal S. aureuscolonization (Table 1). A prevalence of 36.4% of positive isolation were obtained from 31 family members of 20 patients, significantly higher (P=0.0036) than the nonatopic adults examined as controls (Table 1). In healthy children, S. aureuswas isolated at a significantly lower frequency (18 positive out of 90, 20%, P<0.0001) from nasal swabs. Conversely, the frequency of detection from specimens collected from normal skin areas in patients with AD was low (4/117) and did not significantly differ from that of healthy children (0/90). Considering the age, the rate of positive isolation (including either skin or nares) was significantly higher in the group of children aged more than six (P=0.0062) (Table 2). Regarding the impact on disease severity, the presence of S. aureuscolonization appeared significantly related to a more severe clinical expression of pediatric AD (P=0.0001) (Table 3).Table 1
Prevalence ofS. aureus colonization in patients, family members, and controls. Contingency analysis and Fisher’s exact test: *P<0.0001; **P=0.0036.
SubjectsPos/allLesional skin and naresNaresUninvolved skinAtopic children66/117* (57%)47/117 (40.2%)19/117 (16.2%)4/117 (3.4%)Healthy children18/90 (20%)—18/900/90Parents/relatives31/85** (36.4%)—31/850/85Healthy adults16/80 (20%)—16/800/80Table 2
Prevalence ofS. aureusisolates in children with AD according to age.
Age (yrs)Pos/neg (n)Positive (%)<327/2254.13–613/2138.26–1226/875.7*Chi-square testP=0.0062.Table 3
Prevalence ofS. aureusisolates in children with AD according to disease severity.
Disease scorePos/negPositive (%)Low3/1715.0Medium25/2352.0High38/1177.5*Chi-square testP=0.0001Taking into consideration the expression of virulence factors byS. aureus isolates, the prevalence of MRSA strains among the total number of individual isolates in atopic patients was 7.9%, being present in the nose (4.5%) and, more frequently, in the skin lesion (12.8%). According to the molecular analysis, S. aureusstrains isolated from atopic children (a total of 119, of which 96 were from skin lesion and nares of the same patients, 19 from nares of skin negative patients and 4 from healthy skin areas of 4 skin and nare positive patients), 94 (79%) had one or more genes coding for virulence factors such as adhesins, enterotoxins, TSST-1, CAP 5/8, the PV leukocydin, or the capacity to form biofilm (Table 4). Significant associations were found between the severity of the disease and high biofilm producing strains or TSST-1 positivity (P=0.0003 and P=0.002, resp.) (Table 5).Table 4
Prevalence ofS. aureustoxigenic strains in atopic and healthy children.
Toxigenic strains  Pos/all (%)MRSA   Positive (%)AD lesional skin (47)37 (79%)6 (12.8)AD uninvolved skin (4)3 (75%)AD nares (66)51 (77%)3 (4.5)CTRLs nares (18)5 (28%)*0/18*Chi-square test *P<0.0001Table 5
Molecular characterization of bacterial virulence factors ofS. aureus strains isolated in children with AD and disease severity.
Disease ScoreBiofilmAdhesinsEnterotoxinsPVLTSST-1CAP 5/8Low/HighPos/NegPos/NegPos/NegPos/NegPos/NegLow/Medium19/1532/026/61/3112/2034/0High8/4358/045/131/5728/3151/0Fisher’st testP=0.0003P=0.63P=0.18P=0.002To evaluate the possible transmission between nares and skin of the same patient or between patients and their cohabitants, the genetic characterization of strains was assessed by DNA restriction fragmentation by PFGE. Similar PFGE patterns were exhibited by strains collected from skin or nares in the same patient in all 47 positive cases, as reported in Figure1. The PFGE analysis also showed a close similarity among patients and their cohabitants in 75% of cases. Interestingly, a striking correspondence was found between strains isolated from mothers and atopic children (72%), while fathers and brothers showed a lower extent of identity (53% and 58%, resp.). The data from DNA analysis were confirmed by bacterial phenotyping according to the profile of toxins released in the supernatants of S. aureus cultures as detected by the immunoassay (Table 6). The data showed a correlation between the individual patient and at least one of the family members. Considering the expression of drug resistance, the results indicated that strains isolated from parents had a more complex profile of drug resistance than the similar strains isolated from their children (Table 6).Table 6
Immunological detection of toxin array and drug resistance ofS. aureus isolates from patients and their family members.
Source of isolateLocalizationEnterotoxins  TSST-1Antibiotic resistancePatient 6Lesional skinSECGM, PNoseSECPPatient 6, fatherNoseSECPPatient 6, motherNoseSECPPatient 10Lesional skinSECPPatient 10, fatherNoseSECPPatient 15Lesional skinSEBOX, P, ENoseSEBPHealthy skinSEBOX, CM, MUP, RFPatient 15, motherNoseSEBP, CM,Patient 16Lesional skinSEA, TSST-1P, E, MUPNoseSEA, TSST-1P, E, MUPHealthy skinSEA, TSST-1P, E, MUPPatient 16, fatherNoseSEA, TSST-1OX, P, E, MUP, MXF,CM, TEC, TE, FAPatient 19NosePPatient 19, motherNosePPatient 21Lesional skinFFNoseTePatient 21, fatherNoseSEA, SEDPPatient 21, motherNoseSEA, SEDPPatient 23Lesional skinPNosePPatient 23, fatherNoseP, CM, EPatient 23, motherNosePPatient 25Lesional skinPatient 25, motherNosePatient 26Lesional skinP, FFNoseTSST-1PHealthy skinP, FFPatient 26, fatherNoseP, FFPatient 26, motherNoseTSST-1P, E, CMPatient 26, brotherNoseTSST-1P, E, CMPatient 27Lesional skinSEBP, E, CMNoseSEBP, E, CMPatient 27, motherNoseSEBP, E, CMPatient 27, brotherNoseSEBP, E, CMPatient 28Lesional skinPatient 28, fatherNoseP, MUPPatient 28, motherNoseTSST-1Patient 29Lesional skinTSST-1PPatient 29, motherNoseTSST-1PPatient 30Lesional skinTSST-1PNosePPatient 30, motherNoseTSST-1PPatient 31NoseSECP, E, CMPatient 31, motherNoseSECP, E, CMPatient 32NosePatient 32, fatherNoseTSST-1PPatient 32, motherNoseTSST-1PPatient 33NoseTSST-1PPatient 33, motherNoseTSST-1PPatient 38Lesional skinPNosePPatient 38, fatherNosePPatient 38, sisterNosePPatient 44Lesional skinSECP, E, CM, FFNoseSECP, E, CMHealthy skinSECP, E, CMPatient 44, fatherNoseSECPatient 44, motherNoseSECP, E, CMPatient 45NoseTSST-1PPatient 45, motherNoseTSST-1PPatient 58Lesional skinPNosePPatient 58, fatherNosePPatient 58, sisterNosePAbbreviations: P: Benzylpenicillin; OX: Oxacillin; CM: Clindamycin; E: Erythromycin; FF: Fosfomycin and nitrofurantoin; FA: Fusidic Acid; GM: Gentamicin; MXF: Moxifloxacin; MUP: Mupirocin; RF: Rifampicin; TEC: Teicoplanin; TE: Tetracycline.Figure 1
Pulse field gel electrophoresis (PFGE) of different isolates ofS. aureus; the first and last lanes correspond to the DNA ladder; lanes 1–4 correspond to strains collected from lesional skin (1), nose (2), healthy skin (3) of an atopic patient (patient 16), and his father (4); lanes 7–12 show similar strains isolated from lesional skin, nose, healthy skin of an atopic patient (patient 26) and related mother, father, and brother.


## 5. Discussion

Although the etiology of atopic eczema remains unknown, evidence suggests that both genetic and environmental factors play a role in determining both the susceptibility and the severity of the disease [5, 26]. Recent evidence suggests that S. aureus may play a key role in the pathogenesis of AD [8]. In fact, while S. aureusis rarely found on the skin of healthy subjects, as confirmed by our data on 80 control children, is very frequently found in patients suffering of atopic eczema. Several possible pathogenic mechanisms have been suggested for the role of S. aureusinfection/colonization in AD pathogenesis pointing at either a direct chemical irritation or a nonspecific reaction of the S. aureus protein A with immune cells [27] as well as possible mechanisms involving superantigens production [28], which, in turn, exacerbates or maintain skin inflammation in atopic eczema. Indeed, S. aureus produces a group of toxins, which are capable of stimulating large populations of T-lymphocytes, even at distance from the eczematous sites, thus sustaining the activation of the immune system and the persistence of existing lesions. Conversely, the use of oral antibiotics as well as of topical antibiotics/antiseptics in combination with a topical steroid, antifungal agents or antiseptic bath additives, are often effective for the clinical management of AD and help reduce the severity of eczema and at improving the quality of life.In the present study, we characterized the strains ofS. aureus isolated from skin lesion and anterior nares of children with atopic dermatitis and from their cohabitants. The same analysis was performed in healthy subjects either children or adults. Our findings showed that the incidence of S. aureus infection/colonization is higher in children with AD, as compared to matched healthy controls (P<0.0001) (Table 1). These data are in agreement with the prevalence previously found by others (50%−64.2%) [29, 30]. Interestingly, the prevalence ofS. aureus colonization was higher in the patient’s relatives (36%) (P=0.0036) than that found in adult healthy subjects (Table 1). In fact, S. aureus isolates obtained from different sites of the same patients had a close biologic and molecular identity with those collected from patients’ cohabitants (P<0.0001), indicating that they harbored the same S. aureus strain. A reason for such a high rate of S. aureus colonization among the cohabitants of pediatric patients should be probably due to the close physical contact occurring between children and parents/relatives, especially required for care giving. This may concur in establishing a source for reinfection among family members as well as other cohabitants and may sustain intrafamiliar spreading with persistence or reactivation of the disease [30–32]. Considering the expression of drug resistance, results showed in Table 6 indicated that strains isolated from parents had a more complex profile of drug resistance than the similar strains isolated from their children. This could be probably due to an increased exposure to antibiotics given both systemically and locally in adults during their lives. On the other hand, we also found that the presence of S. aureus was associated with an age under three yrs or more than six yrs (P=0.0062) (Table 2), and with a more marked severity of the disease (Table 3). It could be hypothesized that the exposure during the neonatal period is due to the close contact with their caregivers, while older children more frequently interact with the external environment. The assessment of the bacterial genotypic and phenotypic profiles, through immunologic and molecular methods, showed a significant higher frequency of S. aureus strains expressing molecules associated with virulence, such as enterotoxins, adhesins, PVL, biofilm production, or MRSA in children with AD as compared to asymptomatic carriers. Among these biologic properties, the ability to generate biofilm or TSST-1 were significantly more frequent (P=0.0003 and P=0.002, resp.) in patients with a more severe form of the disease as compared to those with a mild disease expression according to the criteria described by Hanifin and Rajka [20]. These factors may thus have an important impact on the clinical outcome and disease severity.


## 6. Conclusions

Our data suggest that the nasal cavity represent a key source forS. aureus skin colonization and that the familiar environment may play an important role in S. aureus colonization. Therefore, the eradication of these reservoirs might have a key impact on both the frequency and outcome of AD [33]. These results may indicate that clinical management of AD should include a routine testing for S. aureusnot only on skin lesion but also in other important body reservoirs such as the nasal cavity [31] and emphasizes the need of performing susceptibility tests for antimicrobial drugs before initiating the therapy. In addition, routine testing should include also the family members of AD patients in order to break the chain of transmission through the establishment of an appropriate antibacterial therapy.



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