Aim To determine whether intestinal Staphylococcus spp. and their pathogenic features differed between coeliac disease (CD) patients and healthy controls.
Methods 60 children, including active CD (n=20) and non-active CD (n=20) patients and healthy controls (n=20), were studied. Staphylococci were isolated from faeces and identified by PCR and DNA sequencing. The carriage of virulent genes, including adhesion (atlE and fbe), cell aggregation (icaD), global regulatory (agr and sar) and methicillin-resistant (mecA) genes, was analysed by PCR.
Results Staphylococcus epidermidis was more abundant in the microbiota of active and non-active CD patients than in controls. Staphylococcus haemolyticus was more abundant in active CD patients than in control subjects. Staphylococcus aureus was less abundant in active CD patients than in the other child groups. Staphylococcus spp. diversity was higher in active CD patients than in non-active CD patients and controls. The presence of the mecA gene and the simultaneous presence of both the mecA and atlE genes were higher in S. epidermidis clones isolated from CD patients, with active and non-active disease, than in those from control subjects. The individual presence of the other virulent genes was lower in S. epidermidis from active CD patients than in those from the other -child- groups.
Conclusions Increased abundance of S. epidermidis carrying the mecA gene, in active and non-active CD patients, most likely reflects increased exposure of these subjects to opportunistic pathogens and antimicrobials.
- Coeliac disease
- gut microbiota
- methicillin resistance
- clinical infectious diseases
- infectious intestinal disease
- inflammatory bowel disease
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- Coeliac disease
- gut microbiota
- methicillin resistance
- clinical infectious diseases
- infectious intestinal disease
- inflammatory bowel disease
Coeliac disease (CD) is an autoimmune enteropathy caused by a permanent intolerance to cereal gluten proteins (gliadins).1 Currently, the only available therapy for CD is the adherence to a strict, lifelong, gluten-free diet; however, the compliance with this dietary recommendation is complex and other alternative strategies are needed.2
Scientific evidence suggests that environmental factors, other than gluten, may play a role in CD pathogenesis. It has been suggested that early infections may increase the risk of suffering CD in susceptible individuals.3 ,4 In addition, imbalances in the gut microbiota of CD patients5 ,6 and infants at genetic risk of CD7 have been reported. Greater numbers of Staphylococcus spp. have been found in faecal and biopsy samples of CD patients, suggesting it may be related with the active phase of CD.8
Staphylococcus spp. are widespread in various environments, including skin and mucosal membranes of humans and many animals, and some species are clinically important because they cause chronic infections.9 For instance, Staphylococcus epidermidis is the most frequent causative agent of nosocomial infections.10 The pathogenesis of staphylococci and, particularly, of S. epidermidis, depends on the presence of genes coding for virulent factors mainly related to biofilm production and antibiotic resistance. The bacterial biofilm is produced in two steps comprising the initial bacterial attachment to the surface followed by a second stage consisting of bacterial proliferation, intercellular adhesion (cell aggregation) and production of an extracellular slimy substance. The AtlE, a cell surface protein exhibiting vitronectin-binding activity is one of the most important factors involved in the primary attachment of S. epidermidis.11 ,12 S. epidermidis can also bind to other extracellular matrix-related proteins, like fibrinogen, via fibrinogen-binding proteins codified by the fbe gene.13 Cell aggregation is mediated by the products of the chromosomal ica locus, which comprises four intercellular adhesion genes (icaA, icaB, icaC and icaD). The ica operon encodes enzymes for the biosynthesis of PIA (polysaccharide intercellular adhesin) leading to multilayer biofilm development.12 ,14 PIA is also important for haemagglutination of erythrocytes by S. epidermidis.15
Quorum-sensing and global regulatory systems also play an important role in the regulation of virulent factors. The genes of two global regulatory loci, agr and sar seem to be involved in the regulation of the virulent factors of S. epidermidis.16 ,17 S. epidermidis is considered an emergent pathogen causing increasing numbers of human infections, probably because a methicillin-resistant gene complex is widespread among isolates of this species.9 ,18 Methicillin resistance is due to the expression of a modified penicillin-binding protein PBP2a (PBP2′) encoded by the mecA gene and located on the mobile element staphylococcal cassette chromosome mec (SCCmec), a genomic island.9
The purpose of this study was to analyse the diversity and species composition of clones of the genus Staphylococcus, isolated from faecal samples of active and non-active CD patients and age-matched controls. Furthermore, the presence of well-known virulent genes was examined in order to establish a possible link between the pathogenic potential of Staphylococcus spp. and CD.
Subjects and sampling
A total of 60 children were included in this study: (1) active CD patients (n=20), who were on a normal gluten-containing diet, showed clinical symptoms of the disease, positive CD serology markers and severe enteropathy, classified as type 3 according to the Marsh classification of CD by duodenal biopsy examination19; (2) patients with non-active CD (n=20), who had been on a gluten-free diet for at least 2 years, showed negative CD serology markers and normal mucosa, or infiltrative lesions classified as type 0–1 and absence of disease symptoms; and (3) healthy control subjects (n=20) without known food intolerance. None of the children in the trial had been treated with antibiotics for at least 1 month prior to sampling. Clinical and paraclinical characteristics of the children are shown in table 1.
Faecal samples from the trial subjects were kept under anaerobic conditions (AnaeroGen, Oxoid, Hampshire, UK) and analysed within 12 h. Aliquots (2 g wet weight) were 10-fold diluted in phosphate-buffered saline (PBS, 130 mM sodium chloride, 10 mM sodium phosphate, (pH 7.2)), homogenised, plated on Baird Parker agar (Scharlau, Barcelona, Spain) and incubated under aerobic conditions at 37°C for 48 h.
Identification of bacterial isolates
Five individual colonies from the highest dilution plate of each subject were randomly selected and grown in Brain Heart Infusion broth (BHI, Scharlau®). Colony and cellular morphology under Gram staining, and DNAse and coagulase activities were evaluated. Species-level identification was carried out by PCR with the primers listed in table 2. Isolates were identified as Staphylococcus aureus or S. epidermidis by a multiplex PCR based on the dnaJ gene (i).20 Other isolated species were identified by sequencing of an 883 bp fragment, amplified with dnaJ degenerate primers (ii),21 or by 16SrDNA partial sequencing (iii).22
Amplicons were purified using GFXtm PCR DNA Kit (GE Healthcare, Buckinghamshire, UK) and sequenced with an ABI PRISM-3130XL Gene Analyzer (Applied Biosystems, California, USA). Search analyses to determine the closest relatives of the retrieved gene sequences were conducted in GenBank using the Basic Local Alignment Search Tool algorithm. Sequences with more than 97% similarity were considered to be of the same species.
Pathogenicity genes of S. epidermidis strains
The presence of fbe, atlE, icaD, agrA, sarA and mecA genes was determined in S. epidermidis isolates (table 2). Multiplex PCR was performed for combined amplification of (iv) atlE, fbe and icaD genes20; and (v) for that of agrA and sarA genes23; and a simplex PCR (vi) was performed for mecA gene amplification.23
The haemolytic activity of the isolates was determined on Columbia agar supplemented with 5% horse blood (COH, BioMerieux). A positive-biofilm phenotype of S. epidermidis strains was determined using Congo Red agar assay.24
Shannon-Wiener diversity index (H') was used to determine the diversity of taxa present in faecal samples of active and non-active CD patients and control children, using PAlaeontological STatistics software.
Differences in Staphylococcus spp. abundance and in the presence of virulent genes were established by applying χ2 test and, when appropriate, by applying the two-tailed Fisher's exact test. Analyses were carried out with Statgraphics software (Manugistics, Rockville, Maryland, USA).
Diversity of Staphylococcus spp. in CD patients and control children
A total of 300 isolates were randomly isolated from faecal samples of the children under study. The abundance of Staphylococcus-like species recovered from active and non-active CD patients and control subjects is shown in table 3.
S. epidermidis isolates were more frequently recovered from active and non-active CD patients than from control subjects (p<0.01 and p=0.03, respectively). Furthermore, Staphylococcus haemolyticus was more common in active CD patients than in controls (p<0.04). In contrast, S. aureus isolates were less frequently recovered from active CD patients than from non-active CD patients and controls (p<0.01). The percentage of DNAse and coagulase-positive S. aureus isolates was similar in all three groups of children. Staphylococcus warneri was less frequently isolated in non-active CD patients than in active CD patients and controls (p<0.01). The diversity of staphylococcus-like spp. was calculated applying the Shannon-Wiener diversity index (H'), which considers both the number and evenness of species. Active CD patients showed higher species diversity (1.71) than non-active CD patients (1.45) and controls (1.44).
Interestingly, 72 isolates were recovered from Braid Parker agar and identified as Enterococcus faecalis or Enterococcus faecium (table 2). The abundance of isolates belonging to the species E. faecium was higher in control children than in CD patients with either active disease or non-active disease. Enterococcus spp. were less frequently isolated in active CD patients (14%) than in non-active CD (26%, p=0.05) and in controls (32%, p<0.01).
Characterisation of S. epidermidis isolates
A total of 75 S. epidermidis isolates were identified in the children under study, and the presence of virulence-associated genes is shown in table 4.
Both the adhesion-encoding genes (atlE and fbe) were less frequently detected in S. epidermidis isolates recovered from active CD patients than in those recovered from non-active CD patients (p<0.01) and controls (p<0.01). The cell-aggregation gene icaD, was also less frequently detected in isolates recovered from active CD patients than in those from non-active CD patients (p<0.01) and controls (p=0.02). There was a good correlation between the presence of this gene and the results obtained with the Congo Red agar assay (data not shown), which determines biofilm production potential. No differences were detected between the haemolytic activities in S. epidermidis strains isolated from the analysed groups of children (data not shown).
The virulent genes corresponding to the global regulatory loci agr and sar were less common in S. epidermidis isolates from active CD patients than in those from controls (p=0.01 and p=0.03, respectively) and non-active CD patients (p<0.01 and p<0.01, respectively). In contrast, the methicillin-resistant gene (mecA) was most frequently detected in S. epidermidis isolates from active (p<0.01) and non-active CD patients (p<0.01) than in those from controls. The isolates carrying both the atlE and the mecA genes simultaneously were also more abundant in active and non-active CD patients (p<0.01) than in controls.
Staphylococcus spp. are important inhabitants of the human skin and mucosa25 and also of the intestinal tract.26 Various studies have reported increased numbers of Staphylococcus spp. in the gut microbiota of active CD patients,5 ,8 which is similar in infants with inflammatory bowel disease (IBD)27 ,28 or allergy.29 ,30 This would suggest there is a relationship between this bacterial group and immune-mediated pathologies. Herein, we show that CD is associated with alterations in species diversity and composition of the faecal Staphylococcus population. S. epidermidis was more abundant in CD patients, regardless of the disease stage, than in controls. Meanwhile, S. haemolyticus was more abundant in active CD patients than in controls. It is known that both species are involved in human infections.31
S. epidermidis is currently considered as an important opportunistic pathogen, especially in healthcare-associated infections.10 ,32 S. epidermidis strains are well equipped with determinants promoting persistence in specific ecosystems, related with the evasion of host defences and biofilm formation.12 Cell surface proteins could contribute to the attachment of S. epidermidis to host cells and tissues,33 ,34 which can increase their pathogenicity. In this study, the individual presence of genes related to biofilm formation (atlE, fbe and icaD) was more common in S. epidermidis isolates from non-active CD patients and controls, which could lead to a more stable colonisation. In fact, biofilm formation is a common colonisation mechanism of both commensal and pathogenic bacteria, facilitating their persistence in different environments, including the gastrointestinal tract35 ,36; this phenomenon has also been related to microbial alterations in IBDs.37
Our study also reports there is an increased abundance of methicillin-resistant S. epidermidis isolates associated with active and non-active CD patients. Antibiotic resistance, and particularly methicillin resistance, is a widespread characteristic of S. epidermidis strains. Methicillin is an antibiotic of first choice against staphylococcal infections, and other antibiotic-resistant genes are more often found in methicillin-resistant than in methicillin-susceptible strains.38 Furthermore, most S. epidermidis infections are hospital-acquired or healthcare related, and methicillin-resistant strains are commonly found in these patients. Hospital visits are frequent before CD diagnosis39 and severe infections are commonly associated with CD,40 ,41 which could explain the increased abundance of methicillin-resistant S. epidermidis in our cohort of CD patients. Hospitalized IBD patients are also at an increased risk of methicillin-resistant S. aureus infections compared with non-IBD patients.28 The simultaneous presence of both adhesin-encoding genes, which could confer increased biofilm formation capacity, and antibiotic-resistant genes in S. epidermidis isolates from CD patients under study, may explain the persistence of this species in these patients even after the administration of a long-term gluten-free diet.42 ,43
Unlike S. epidermidis, the abundance of S. aureus was higher in healthy children than in active CD patients. Consistent with this, it has previously been reported that S. aureus is often present in stools of healthy infants44; it was suggested that parental skin S. aureus strains can easily establish in the infant gut probably due to poor competition from other gut bacteria. Similarly, it could be speculated that the increased abundance of S. epidermidis and the increased diversity in Staphylococcus spp. is associated with the reduced abundance of S. aureus in active CD patients due to higher competition among species of the same genus for the same ecological niche.
This study demonstrates that CD is associated with shifts in Staphylococcus species diversity and abundance in the intestinal microbiota. S. epidermidis isolates carrying the mecA gene, and both the mecA and atlE genes, were more abundant in CD patients than in controls. This suggests that children with CD have greater exposure to opportunistic staphylococcal pathogens and antimicrobials, which in turn affects the composition/features of their intestinal microbiota.
Staphylococcus epidermidis isolates carrying the mecA gene, which confers resistance to methicillin, are more abundant in CD patients than in controls.
This finding suggests that children with CD have greater exposure to opportunistic staphylococcal pathogens and antimicrobials.
The contract (PTR95-0987.OP.01 from MICINN and scholarship (Institute Danone) to E. Sánchez are fully acknowledged.
Funding This work was supported by Grants AGL2008-01440/ALI, and Consolider Fun-C-Food CSD2007-00063 from the Spanish Ministry of Science and Innovation (MICINN).
Competing interests None.
Patient consent Obtained.
Ethics approval Ethics approval was obtained from the Ethics Committee of Consejo Superior de Investigaciones Científicas (CSIC) and the hospitals taking part in the study (Hospital Universitario La Fe and Hospital General Universitario, Valencia, Spain). Children were enrolled in the study after written informed consent was obtained from their parents.
Provenance and peer review Not commissioned; externally peer reviewed.
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