Methicillin-resistant Staphylococcus aureus from infected skin lesions present several virulence genes and are associated with the CC30 in Brazilian children with atopic dermatitis

ABSTRACT Atopic dermatitis (AD) is a chronic inflammatory skin disease and colonization by Staphylococcus aureus may affect up to 100% of these patients. Virulent and resistant isolates can worsen AD patient clinical condition and jeopardize the treatment. We aimed to detect virulence genes and to evaluate the biofilm production of S. aureus isolates from infected skin lesions of children with AD. Methicillin resistance was detected by phenotypic and molecular tests and the virulence genes were detected by PCR. Biofilm formation was assessed by bacterial growing on microtiter plates and later stained with safranin. Genotyping was performed by Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing. Among 106 AD patients, 55 (51.8%) had developed S. aureus cutaneous infections and 23 (41.6%) were methicillin-resistant (MRSA). All 55 isolates carried the fnbA, hla, icaA, sasG, and seu genes, and more than 70% presented cna, eap, ebpS, hlg, and pvl genes. Clonal complex (CC) 30 was the main lineage found (34.5%), especially among MRSA isolates (52.2%). The egc cluster and the bbp gene were significantly the most frequent in MRSA isolates and in USA1100/ST30/CC30 lineage. Most of the isolates (74.5%) were non-biofilm producers and many of them only started to produce it in the presence of fibrinogen. There was no significant association between S. aureus isolates features and the AD severity. This study demonstrated a high frequency of CC30 MRSA isolates presenting several virulence genes in infected skin lesions of AD children in Brazil, that may influence the severity of the disease and the treatments required.


Introduction
Atopic dermatitis (AD) is a chronic skin disorder which the main signs and symptoms are pruritus, xerosis, and eczematous lesions placed on hands, neck, popliteal, and antecubital fossae [1]. The severity of AD can be classified by the SCORAD (scoring atopic dermatitis) index, which is based on lesions and symptoms extension and intensity, and can be classified as mild (SCORAD <25), moderate  or severe (>50) [2]. Genetic predisposition, skin barrier disruption, exposure to environmental factors, and dysbiosis seem to be associated with the disease [3]. Skin colonization by S. aureus may contribute to the onset and/or aggravation of these lesions [4]. Prevalence of this pathogen in AD skin lesions may affect up to 100% of these patients [5,6] and consequently, S. aureus infections are the most common aggravating factor of AD. Nevertheless, molecular epidemiology of S. aureus from infected AD lesions, especially in developing countries, remains unknown.
Exacerbations of AD caused by S. aureus are probably due to staphylococcal toxins that can aggravate the eczema [7]. Furthermore, staphylococcal superantigens (SAgs) have been shown to rapidly induce the IL31 mRNA expression in the skin of atopic subjects in vivo and in peripheral blood mononuclear cells in vitro, suggesting that chronic colonization and super infection by S. aureus can contribute to pruritus and inflammatory changes in patients with AD [8].
S. aureus presents a great number of virulence factors, including secreted proteins and enzymes that might be used to establish and maintain the infection [9]. Some studies have evaluated isolates recovered from skin of AD patients and found genes of SAgs such as the toxic shock syndrome toxin-1 (TSST-1), staphylococcal enterotoxins (SEs), and the SE-like proteins [10][11][12][13]. Other exotoxins described in S. aureus isolates from AD patients include: cytolysins, particularly the α-, β-, γ-, and δ-toxins; phenol soluble modulins and leukocidins [9]. Also, some S. aureus isolates from AD skin lesions have been shown to produce biofilm, which could be associated with inflammation and pruritus probably due to the occlusion of sweat ducts [14].
As previous explained, S. aureus colonization can aggravate AD and the virulence factors produced by this pathogen can play a significant role in disease manifestations. Therefore, the aim of this study was to assess the characteristics associated with virulence, antimicrobial resistance, and clonality of the S. aureus isolates from infected skin lesions of Brazilian children with AD, and to clarify the relationship of these characteristics with the severity of the disease.

Clinical isolates, setting and ethics statement
A cross-sectional study was conducted, between September 2011 and September 2013, at the pediatric dermatology outpatient clinic of the Instituto de Puericultura e Pediatria Martagão Gesteira (IPPMG) of the Universidade Federal do Rio de Janeiro (UFRJ), Brazil, where 130 AD pediatric patients were being accompanied at the time attended. The target population of the study included male or female patients diagnosed with AD who were 15 years old or less. The study was approved by the ethical committee of the IPPMG (Nº 51/11).
Swabs of skin infected lesions were obtained from all patients clinically diagnosed by pediatric dermatologists [15]. In brief, a sterile swab moistened in saline solution (0,85%) was placed on the skin injury, rotated three times, and plated onto mannitol salt agar (Oxoid; Basingstoke, United Kingdom). The plates were incubated for 48 h at 35°C and the bacterial isolates were characterized by standardized tests [16].

Methicillin susceptibility tests and SCCmec typing
Methicillin susceptibility was determined by disk diffusion using the 30 µg cefoxitin disk (CECON; São Paulo, SP, Brazil) according to Clinical Laboratory Standard Institute recommendation (CLSI, 2014). The Staphylococcus aureus ATCC 25923 was used as control.
Bacterial DNA was extracted by the method described earlier using guanine isothiocyanate [17] and all MRSA isolates were subjected to SCCmec typing, according to the method described by Kondo et al. [18].
The primers HLA-1 (5ʹ-AATCCTGTCGCTAATGCC) and HLA-2 (5ʹ-CAGCAATGGTACCTTTCG) used in the PCR for the hla gene were designed previously by our group [19] and generated a fragment of 208 pb. Amplification conditions were: denaturation at 94°C for 2 min, followed by 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min, with a final extension at 72°C for 5 min. The expression of the hld gene was assessed by measuring the hemolysin activity in blood agar, as described by Harigaya et al. [20]. The RN4220 strain was used as control.

Biofilm formation
The biofilm formation was evaluated for all isolates on 96-well microtiter polystyrene plates TPP 92,096 (Techno Plastic Products; Trasadingen, Switzerland) as described by Ferreira et al. [21]. Twenty microliters of a bacterial suspension in sterile distilled water corresponding to the 0.5 McFarland standard were added in triplicate to the wells containing 180 µl of tryptic soy broth, TSB (Becton, Dickinson and Company; Sparks, MD, USA), supplemented with 1% glucose (Isofar; Duque de Caxias, RJ, Brazil). The plates were incubated at 37°C for 24 h without shaking. The biofilm was heatfixed at 60°C for 1 h, stained with 0.1% (w/v) safranin for 15 min, and distained with 95% (v/v) ethanol for 30 min. The biofilm phenotype was categorized as absent, weak, moderate, or strong according to Stepanović et al [22]. Besides that, 13 isolates were randomly chosen in order to investigate biofilm production in wells coated with 50 µg/ml of human plasmatic fibrinogen (Sigma Chemical Company; St. Louis, MO, USA) [23]. All tests were performed in triplicate. Staphylococcus aureus ATCC 33591 was used as a control for biofilm formation.

Genotyping tests
Pulsed-field gel electrophoresis (PFGE) was carried out for all S. aureus isolates, after digesting genomic DNA with SmaI (New England Biolabs, Inc.; Beverly, MA, USA) by the method described previously [24] with pulse times increasing from 1 to 35 seconds and a running time of 23 hours. The Dice index and the unweighted pair group method with arithmetic average (UPGMA) with 0.5% optimization and 1% position tolerance were used for similarity and cluster analysis. After PFGE, isolates were grouped in genotypes according to their similarities in band patterns [25] and the clonality was obtained by comparisons with previously published pictures [26]. Multilocus sequence typing (MLST) [27] was performed for one representative isolate of each genotype to determine the sequence type (ST). Internal fragments of seven housekeeping genes have been amplified (arcC, encoding carbamate kinase; aroE, shikimate dehydrogenase; glpF, glycerol kinase; gmk, guanylate kinase; pta, phosphate acetyltransferase; tpi, triosephosphate isomerase; and yqiL, acetyl coenzyme A). The allele sequences were analyzed using Bioedit 7.0 software and the MLST database (www. pubmlst.gov). In this technique, each allelle sequence is assigned with a number. The sequence type (ST) (or allelic profile) is based on the combination of the seven assigned numbers [28]. Minimum spanning trees (MST) were created by goeBURST implemented in PHYLOViZ [29] 2.0 software. The STs were represented by circles; the size of a circle is proportional to the number of isolates of this particular ST.

Statistical analysis
Virulence genes and clonality data were analyzed using SPSS (IBM; Armonk, NY, USA) 21.0 software program for Windows. The Exact Fisher and Chi-Square tests were used to analyze the data. The biofilm data were analyzed using the GraphPad Prism 6.01 program (San Diego, California, USA). The two-way Anova test followed by the Sidak test for multiple comparisons was used in order to evaluate the biofilm formation in the presence of fibrinogen. Significance was established at 5% (p < 0.05). Unadjusted associations between independent variables were assessed through odds ratio (OR) and 95% confidence interval (CI).

Clinical data
During the period of this study, 106 pediatric patients with AD were attended in IPPMG ambulatory. Among these, 55 (51.9%) presented cutaneous infection caused by S. aureus. The clinical data were assessed for 49 patients and revealed that 67% were female. The median age of subjects was 7 years and 29 of the patients (59.2%) presented moderate AD, 11 (22.4%) had mild AD and 9 (18.4%) presented the severe form of the disease. The SCORAD data for 6 (10.9%) patients was not available.

Methicillin resistance and genotypes
Among the 55 S. aureus isolates, 23 (41.8%) were MRSA and 32 (58.2%) were methicillin-susceptible S. aureus (MSSA) (  Table 2). One isolate was not related to the MRSA endemic clones and the MLST method showed the CC97. MRSA isolates were found in 45.5%, 41.4% and 33.3% of patients with mild, moderate, and severe AD, respectively, and the occurrence of virulence genes and genetic lineages was balanced among isolates recovered from these patients.

Biofilm production
The most part of the 55 S. aureus isolates investigated for biofilm formation were categorized as nonproducers (41; 74.5%), while 7 (12.7%) were weak producers and 6 (10.9%) moderate producers. Only one isolate was considered a strong biofilm producer ( Figure 2a). Interestingly, all the 13 isolates randomly selected with variable profiles of biofilm production presented a significant increase in biofilm formation or only started to produce biofilm when evaluated in wells coated with fibrinogen ( Figure 2b).

Discussion
Staphylococcus aureus remains a major concern for AD pediatric patients. The virulence factors produced by this pathogen, such as adhesins, superantigens (SAgs), and cytotoxins seem to play significant roles in the development and behavior of the disease. Here we investigated the features related to S. aureus isolates from infected skin lesions in Brazilian children with AD, as well as the relationship of these characteristics with the severity of the disease. Although we did not find any significant association between the severity of the disease and presence of virulence genes, methicillin resistance, or clonality, we detected a high frequency of USA1100/ST30/CC30 MRSA isolates carrying a great number of virulence genes, with a significant number of the pvl, bbp and egc cluster genes. These epidemiological aspects have profound relevance and can determine the worsening of the disease and the treatment refractoriness.
In this study, a high rate of methicillin-resistance (41.8%) among the S. aureus isolates from infected lesions of AD patients was found, whereas in USA, Matiz et al. [30] found only 14% of MRSA isolates in this lesion type. When we compare the colonization rates by MRSA between North American children, which reaches up to 25% [31] and the Brazilian children that is about 45% [32] we can suppose that this difference must be due to the geographic characteristics of each country. Regarding the SCCmec type, some authors have showed the predominance of the type IV in MRSA isolates from AD children [5,33]. Interestingly, the SCCmec IV, which was found in all isolates in our study, is prevalent in community MRSA isolates recovered in Brazil [34], showing that S. aureus isolated from AD patients probably follows the same pattern of isolates recovered from the community in each country. Molecular analysis of all S. aureus isolated in the present study showed that 83.6% of them and 95.6% of MRSA isolates belonged to CCs 1, 5, and 30. This predominance was not a surprise, since these are the major lineages of S. aureus found in Brazilian hospitals [35] and community [34]. We also observed the sporadic STs 8, 15, 45, 97, and 398. These findings suggest that isolates from infected lesions may have different origins, not only from widespread lineages but also from the patient's own microbiota or family [36].
The CC30 was notably the most common lineage found in the present study. However, several studies involving S. aureus isolates from the skin of AD patients have shown that CC30 isolates are rare [13,-37-39]. An Irish study from Fleury et al. [38] showed that CC1 was the predominant lineage of S. aureus on the skin of AD children, while CC30 was prevalent in nasal swabs of healthy children. Geoghegan et al. [40] in a study conducted in Ireland hypothesized that an enhanced adhesion to corneocytes could contribute to the success of the CC1 strain, as well as the possibility of CC1 growing better than CC30 strains in the skin environment. In Spain, when Rojo et al. [13] compared S. aureus isolates from AD patients with controls, they found the CC5 lineage as the most prevalent in AD patients, whereas CC30 was the most common lineage among control individuals. Moreover, the analysis of virulence genes showed that isolates from AD patients were notably more virulent when compared to controls. The authors then suggested that the virulence profile could be closely related to the success of a particular lineage in colonize and trigger the infection on atopic skin [13].
Possibly the high frequency of CC30 isolates in our study is related to a great number of virulence factors carried by isolates that belong to this lineage circulating in Brazil. Here, we demonstrate that pvl, sei, sen, seo, and bbp genes were significantly more frequent in CC30 than in CC1 and CC5. The remarkable association between methicillin resistance and high virulence among CC30 isolates in Brazil may be the key to understand a molecular epidemiology of S. aureus recovered from AD in the country. Indeed, CC30 S. aureus isolates from South America, including Brazil [41] and Argentina [42] seem to present more virulence features and resistance to methicillin than isolates from that lineage found in AD patients from Ireland [43] and Spain [44], which would confirm our findings.
All 55 S. aureus isolates of the present study showed multiple virulence genes. The hla, seu, fnbA, icaA, and sasG genes were found in all isolates, hlg were found in 98.2%, and cna in 70.9% of isolates. Aggarwal et al. [45]  found similar rates of hla¸ hlg, icaA, and cna in a study with MSSA and MRSA isolated from a variety of infections in India. However, these authors detected different rates of sea, sec, and sei, which may indicate that some genes are common in S. aureus, regardless of the location of the infections, while others may be more or less associated with infections in atopic skin. Genes of the egc cluster ranged from 43% (sei) to 63.6% (sem) and almost 70% of the isolates presented at least one of these genes. High rates ranging from 65.6% to 71.3% of S. aureus isolates carrying at least one of these genes have been associated with non-infected AD lesions [11][12][13]. In 2004, Holtfreter et al. [46] showed that antibodies for egc-encoding enterotoxins inhibited these toxins with a 10-to 100-fold-reduced potency compared with antibodies specific to the classical enterotoxins, indicating a low efficiency in neutralizing egcencoded superantigens by serum factors. The high rates of these genes found in the present study, and the immunological aspects associated to the enterotoxins egc cluster may indicate a relevant contribution in AD pathogenesis. In the present study, egc cluster and bbp genes were more presented among MRSA isolates than in the MSSA. Although MSSA isolates are usually considered to harbor more virulence genes [47], some authors have already shown that seg and sei genes are significantly more prevalent in MRSA [48]. These findings support the hypothesis that MRSA lineages are evolving over time, acquiring new features, and gaining the ability to trigger infection [49].
The pvl gene was detected in 70.9% of the isolates, a percentage much higher than those observed by other authors in studies involving AD in children. Chiu and colleagues [6] in Singapore and Pascolini and colleagues [50] in Italy evaluated S. aureus isolates from noninfected AD lesions and found 3% and 2.2% of pvlpositive isolates, respectively. Interestingly, among isolates recovered from skin infections in non-atopic patients, the rates of pvl genes can reach 90% [51]. This evidence suggests that the PVL plays a relevant role in cutaneous infections, regardless the patient's health status. However, it is worth to mention that AD patients have chronic wounds that persist for years, making these individuals that are colonized with these strains constantly susceptible to invasive infections.
We did not find any association between a virulence gene or a specific lineage with the severity of AD. Some studies have shown that S. aureus isolates that carry any SAg genes are associated with greater severity of AD [10,52]. However, Sag production was not found exclusively in isolates from patients with AD but also among isolates from other patients, suggesting that other virulence factors may be contributing to the severity of AD [52]. It is possible that AD can be aggravated by the action of one of the toxins or by the potentiation of the action of two or more toxins or even by the presence of other aspects associated with the pathology of AD.
This study observed that 13 isolates that produced little or no biofilm presented a significant increase or only started to produce higher amounts of biofilm when they were grown in wells coated with fibrinogen. The role of the biofilm by S. aureus in AD is still poorly investigated [14,53,54]. Di Domenico et al. [53] showed that S. aureus isolated from skin lesions of AD patients were able to produce high amounts of biofilm, especially the ones isolated from patients with severe AD. Ramundo et al. [54] showed that USA1100/ST30 Brazilian isolates, a prevalent lineage found in the present study, were often considered as weak biofilm producers. In our study, most of the isolates did not produce biofilm even though they presented genes related to its formation. However, in the presence of fibrinogen, a protein that is found in the skin lesions of AD patients due to the scratching caused by constant itching, the biofilm formation occurred. In our study, biofilm was significantly produced by all isolates tested, demonstrating that in vivo this virulence factor could favor the colonization and permanence of this species in the skin.
Although all patients enrolled in the study attended the AD outpatient clinic, only 55 children presented skin infections, and most of them presented moderate AD. The low number of subjects and the unbalanced samples of disease severity can affect the statistical analyses. Although there are differences in microbial community between different parts of the body lesions locations were not evaluate. For example, Staphylococcus is the most common genus in some moist areas, such as plantar heel, popliteal fossae, and occiput region in AD patients [55,[55][56][57][58][59][60][61][62][63][64][65][66][67].
In conclusion, this study showed a high frequency of CC30 MRSA isolates carrying significantly more virulence genes than other lineages, including pvl and egc cluster genes. Moreover, the isolates considered to be non-biofilm producers started to produce this virulence factor in the presence of fibrinogen. Therefore, this study highlights the epidemiological relevance of the data and its influence on the worsening of the disease and on the impairment of the treatment of AD.