Bifidobacterium and Lactobacillus. We used a sera collection consisting of healthy individuals , Crohn's disease patients , and ulcerative colitis patients . Levels of IgA antibodies developed against a cell-wall hydrolase from Lactobacillus casei subsp. rhamnosus GG (CWH) were significantly higher in the IBD group . The specificity of our measurements was confirmed by measuring IgA antibodies developed against the CWH peptide 365-VNTSNQTAAVSAS-377. IBD patients appeared to have different immune response to food bacteria. This paper sets the basis for developing systems for early detection of IBD, based on the association of high levels of antibodies developed against extracellular proteins from food and probiotic bacteria.Inflammatory bowel disease (IBD) is an autoimmune disease characterized by a chronic inflammation of the gastrointestinal tract mucosa and is related to an abnormal immune response to commensal bacteria. Our aim of the present work has been to explore the levels of antibodies (IgG and IgA) raised against extracellular proteins produced by LAB and its association with IBD. We analyzed, by Western-blot and ELISA, the presence of serum antibodies (IgA and IgG) developed against extracellular protein fractions produced by different food bacteria from the genera  Inflammatory bowel disease (IBD) is an autoimmune disease characterized by a chronic inflammation of the gastrointestinal tract (GIT) mucosa. Depending on the severity and location of the injuries, two main forms are distinguished, Crohn's disease (CD) and ulcerative colitis (UC). Both are chronic disorders of unexplained origin, in which persistent ulcerations appear in the small or large bowel mucosa. Interestingly, genetic susceptibility only explains up to 23% of the disease, in the case of CD 16% for UC), with the rest being attributed to environmental factors, such as an exacerbated response of the innate immune system to the commensal microbiota [6% for UC Bacteroides fragilis offers protection from experimental colitis, induced by Helicobacter hepaticus, in an animal model, with this beneficial activity being dependent on the presence of an exopolysaccharide [Experiments in germ-free animals have shown that microbial colonization is crucial in the instruction, maturation, and regulation of the immune system. For instance, the presence ofccharide . In addiccharide . Indeed,ccharide . Faecalibacterium prausnitzii and Escherichia coli in IBD patients resulted in different fecal bile acid compositions, with implications in the perpetuation of chronic inflammation in IBD [ F. prausnitzii [Implications of microbiota dysbiosis extend beyond the obvious differences in microbial composition from a functional and metabolic point of view. For instance, decreased ratios betweenn in IBD . In the n in IBD . For thin in IBD . Althougusnitzii –10. Inteusnitzii , 12. Saccharomyces cerevisiae (ASCA), the outer membrane porin C protein from E. coli (anti-OmpC), or the flagellin from members of the Clostridium cluster XIVa [IBD is associated with antibodies raised against extracellular molecules of GIT microorganisms, such as extracellular mannan fromter XIVa –15. The ter XIVa , 16.Our aim with the present work has been to explore the levels of antibodies (IgG and IgA) raised against extracellular proteins produced by LAB and its association with IBD. The main results are discussed next. Lactobacillus casei subsp. rhamnosus GG (LGG), Lactobacillus acidophilus DSM 20079T, Lactobacillus reuteri DSM 20016T, Bifidobacterium longum subsp. longum NCIMB 8809, Bifidobacterium bifidum LMG 11041T, and Bifidobacterium animalis subsp. lactis IPLA 4549. All strains were grown in MRS  broth  supplemented with 0.05% (w/v) L-cysteine (MRSC) . Agar (1.8% (w/v)) was added to the broth when colony isolation was necessary. In all cases, cultures were incubated in an anaerobic chamber model MG500  with a defined atmosphere composed of 10% (v/v) H2, 10% (v/v) CO2, and 80% N2.Six bacterial strains representing microorganisms used in human nutrition, or as probiotics, were used in this study:μm) and kept for further analysis. MRSC was used as control supernatant.As routine culturing, isolated bacterial colonies were recovered on MRSC agar plates from frozen stocks stored at −80°C (MRSC supplemented with 40% (v/v) glycerol). Single colonies were used for inoculating MRSC tubes, which were kept in the anaerobic chamber ON. Fresh MRSC bottles containing different volumes, depending on the experiment (from 50 mL to 400 mL), were then inoculated from the ON precultures (1% v/v). These cultures were incubated in the same conditions up to early stationary phase of growth (from 12 to 48 h depending on the bacterium). Cultures were then centrifuged , and the supernatants were filtered (0.45 μL of Laemmli buffer 5X [μL were loaded in polyacrylamide gels (12.5%) under denaturing conditions (SDS-PAGE). Prestained Page Ruler  was used as molecular mass marker. Extracellular proteins were separated according to Laemmli [Extracellular proteins were obtained from bacterial supernatants following the protocol described by Sánchez et al. . Brieflyuffer 5X  with the Laemmli , in an eμg of protein was separated in SDS-PAGE as described above. Proteins were transferred and immobilized onto polyvinylidene fluoride (PVDF) membranes  for 30 min under a constant voltage of 50 V. PVDF membranes were blocked with PBS  supplemented with 0.1% (v/v) Tween-20 (PBST) (Sigma-Aldrich) and with 5% (w/v) skimmed milk (PBST-L) (Oxoid Ltd.) for 3 h at room temperature. Membranes were washed twice and individual sera were diluted to 1 : 100 in PBST-L and incubated over the membranes  with slight agitation. Membranes were then washed 4 times with PBST and incubated for 1 h with a secondary antibody conjugated to horseradish peroxidase (horseradish peroxidase-conjugated anti-human IgA or IgG (Sigma-Aldrich)), diluted to 1 : 2,000 in PBST-L. For detection of immunoreactive bands, a commercial solution containing the chromogenic reagents chloronaphthol and diaminobenzidine  was used.For detection of immunoreactive bands within the different extracellular protein extracts, 40 https://sede.isciii.gob.es/). Demographic and clinical parameters of the UC and CD patients are shown in Tables Detection of immunoreactive bands was performed using a collection of sera and consisted of 50 samples from healthy individuals, 37 samples from individuals with CD, and 15 samples from individuals with UC. Approval for the study was obtained from the Regional Ethics Committee for Clinical Investigation  and all determinations were performed with fully informed written consent. This collection is deposited in the “Colección del Registro Nacional de Biobancos”, Ref. C.0001263 , and finally dialyzed against PBS for 24 h at 4°C . Protein concentration was determined by the bicinchoninic acid test , using a series of bovine serum albumin standards.μg of CWH, purified as described above, was dissolved in PBS and coated on the surface of a 96 well plate (Nunc) 1 h at 37°C and then ON at 4°C. Wells were washed twice with 300 μL of PBST and blocked with 200 μL of 1% (w/v) bovine serum albumin in PBS (1 h at RT and with gentle shaking). Wells were washed twice again with 300 μL of PBST and incubated with 200 μL of the sera dilutions, made with PBST, for 2 h at RT. The dilutions tested were 1 : 100, 1 : 250, 1 : 500, 1 : 1,000, 1 : 2,500, 1 : 5,000, and 1 : 10,000. Wells were washed twice with 300 μL of PBST and incubated with 100 μL of a secondary antibody dilution in PBST   in PBST, for 1 h at RT. Finally, wells were washed twice with PBST and 100 μL of the chromogenic substrate ultra-TMB-ELISA (Thermo Fisher Scientific) was added and incubated for 20 min at RT. Reactions were stopped with 2 M H2SO4 and the Abs450 of the wells measured. The titer of each serum was defined as the last dilution of serum that gave a positive reaction (Abs450 threshold = 0.300).Specific IgG and IgA antibodies developed against the CWH of LGG were titrated in all the sera. Two http://blast.ncbi.nlm.nih.gov/Blast.cgi) against the curated, nonredundant “nr” database. Homologous proteins present in other species belonging to the Firmicutes phylum were retrieved and aligned, and the comparative analysis allowed us to identify a specific peptide present in the CWH protein from L. casei subsp. rhamnosus that was not present in other intestinal or food bacteria species . Two mg of the specific CWH peptide (CWHp) was chemically synthesized by the company Genecust (Luxembourg), with a minimum purity of 95%.Homology searches using the CWH amino acid sequence  were performed using the BlastP algorithm implemented at the National Center for Biotechnology Information (μg of the peptide was coated on the surface of 96-well plates (Nunc), and the following serum dilutions were tested for titration: 1 : 100, 1 : 250, 1 : 500, 1 : 1,000, 1 : 2,500, 1 : 5,000, and 1 : 10,000. The rest of the procedure was exactly as described for the CWH.Antibodies developed against the CWHp and present in the following sera C72, C65, C49, C59, P8, P39, P34, and P20 were determined by ELISA as described before. These sera were chosen as being representative for the specific CWH anti-IgA/anti-IgG values. Five In order to evaluate and analyze distribution of data, the following tests were applied: the Runs test, Levène's test, and the Kolmogorov-Smirnov test. The nonparametric Kruskal-Wallis test was used for median comparison between groups . All tests were performed using SPSS v18.0 software.Several immunological studies have shown that the human immune system, in the framework of IBD, reacts differentially against many of the microorganisms composing our gut microbiota by producing specific IgA and IgG responses such as ASCA . ExperimSusceptibility to IBD has been associated with the so-called leaky gut syndrome (LGS), in which a decrease in the gut epithelial barrier led to an increase of antigens from food and bacteria from the gut lumen leaking into the body . Also, i L. casei cell-wall hydrolase, also known as P-75. This is a cell-wall hydrolase able to hydrolyze muropeptides from L. casei [Extracellular proteins from the bacteria strains used in this study, all of them representative of microorganisms used as probiotics or in human nutrition, were immobilized on PVDF membranes and submitted to immunoblot analysis. Incubation with pools of sera of the three different groups , showed the presence of different immunoreactive bands when membranes where developed using an anti-human IgA antibody conjugated with horseradish peroxidase . The preL. casei . In addiL. casei . CWH andL. casei , accomplL. casei , 33.At this point, our results strongly suggested that the levels of specific anti-CWH IgAs were increased in the IBD sera. As Western-blot is very limited for antibody quantification, we purified enough amounts of CWH following the procedure described in the Material and Methods section. Once CWH was purified, and after verifying its purity by SDS-PAGE, the protein was used for the coating of 96-well plates and for quantifying the levels of specific antibodies present in the sera collection. After incubation of the different sera with the coated CWH, we used two types of secondary antibodies for detection of different antibody isotypes specifically developed against CWH, anti-human IgG, and anti-human IgA.13. In this context we can mention ASCA, p-ANCA, and anti-flagellin antibodies, with the latter being even more frequent in other inflammatory disorders such as irritable bowel syndrome (IBS) [While levels of specific IgGs remained constant between the two populations  with the exception of some outlier, levels of anti-CWH IgA were significantly higher in the IBD sera . Howeverme (IBS) . In a stme (IBS) , UC pati L. casei subsp. rhamnosus 365-VNTSNQTAAVSAS-377. Chemical synthesis of the peptide allowed us to measure the specific antibodies (anti-IgG and anti IgA) present in the different sera. In this case, we used the sera corresponding to these healthy controls/IBD patients C49, C59, C65, C72, P8, P20, P34, and P39. These sera were chosen as representative of the specific CWH anti-IgA and anti-IgG titer distribution. IgA titers against the specific CWH peptide, although lower than those measured for the whole CWH protein, were also higher in the IBD sera and lower in the healthy controls (In a step forward, we compared the amino acidic sequence of the CWH protein from LGG against the nonredundant protein database at the NCBI servers, in order to determine whether the anti-CWH antibodies were or were not specific to this protein. A BLAST analysis allowed us to identify a 13-mer peptide which was only present in the CWH fromcontrols . Mycobacterium avium subsp. paratuberculosis, has been proposed as a trigger factor for IBD, although controversial data have been published so far [ M. avium subsp. paratuberculosis in Japanese patients suffering from CD have been reported [Surprisingly, titers corresponding to the specific CWH-peptide IgG antibodies were zero in all the sera, suggesting that they have been developed against homologous surface proteins of other Gram positive bacteria. In this sense, presence of certain enteropathogens, notably infection byd so far . Also, ireported . Bacteroides sp., Parabacteroides sp., E. coli, and Enterococcus sp. were increased [Why levels of IgA antibodies against extracellular proteins produced by gut bacteria are more elevated in IBD patients? It is known that increased gut permeability may correlate with IBD susceptibility/risk. This elevated passage of bacterial antigens in the intestinal mucosa may lead to a concomitant increase in the immune response through specific antibody production for compensating this failure in the barrier function of the gut epithelium . In the ncreased . L. casei are associated with IBD. Quantification of antibodies against extracellular proteins from food and probiotic bacteria may be helpful in the early diagnosis of IBD. Therefore, our results suggest that anti-CWH IgA levels have great potential for being used for early detection of CD and UC; however, validation of these findings in different populations of greater sample size would be required, together with the identification of other extracellular protein targets present in food and probiotic bacteria.Our experiments showed that IgA antibodies specifically developed against an extracellular protein of


Symptom- and sexual history-based testing i.e., testing on indication, for anorectal sexually transmitted infections (STIs) in women is common. Yet, it is unknown whether this strategy is effective. Moreover, little is known about alternative transmission routes i.e. by fingers/toys. This study assesses anorectal STI prevalence and infections missed by current testing practice, thereby informing the optimal control strategy for anorectal STIs in women.Chlamydia trachomatis and Neisseria gonorrhoeae. Data were collected on demographics, sexual behaviour and symptoms. Women were assigned to one of the categories: indication , fingers/toys (only reported use of fingers/toys), or without indication.Women (n = 663) attending our STI-clinic between May 2012-July 2013 were offered routine testing for anorectal and urogenital Of women, 92% (n = 654) participated. There were 203 reports (31.0%) of anal sex and/or symptoms (indication), 48 reports (7.3%) of only using fingers/toys (fingers/toys), and 403 reports (61.6%) of no anal symptoms, no anal sex and no anal use of fingers/toys (without indication). The overall prevalence was 11.2% (73/654) for urogenital chlamydia and 8.4% (55/654) for anorectal chlamydia. Gonorrhoea infections were not observed. Prevalence of anorectal chlamydia was 7.9% (16/203) for women with indication and 8.6% (39/451) for all other women (P = 0.74). Two-thirds (39/55) of anorectal infections were diagnosed in women without indication. Isolated anorectal chlamydia was rare (n = 3): of all women with an anorectal infection, 94.5% (52/55) also had co-occurrence of urogenital chlamydia. Of all women with urogenital chlamydia, 71.2% (52/73) also had anorectal chlamydia.Current selective testing on indication of symptoms and sexual history is not an appropriate control strategy for anorectal chlamydia in women visiting an STI clinic. Routine universal anorectal testing is feasible and may be a possible control strategy in women. Yet costs may be a problem. When more restricted control measures are preferred, possible alternatives include (1) anorectal testing only in women with urogenital chlamydia (problem: treatment delay or loss to follow up), and (2) direct treatment for urogenital chlamydia that is effective for anorectal chlamydia as well. Chlamydia trachomatis (Ct) and Neisseria gonorrhoeae (Ng) are the most prevalent bacterial sexually transmitted infections (STIs) in women in high income countries and have major public health consequences[equences-3. In adequences,4-11 of equences. Howeverequences, i.e., sequences,13. Thisequences. The impequences and can equences,16 and requences,6. MoreoIn the control of anorectal chlamydia there are 2 key stones: first is identification (diagnosis by testing) and second is treatment. In high risk groups there is evidence that many anorectal STI are missed by current testing practice on indication. A study using routine universal anorectal testing in high-risk women found that selective testing on indication misses over half of anorectal infections . It is uAdequate treatment for anorectal chlamydia is currently under debate. Guidelines in the UK and US recommend both single-dose azithromycin and a 7-day course of doxycycline as equal treatments for uncomplicated anorectal chlamydia in non pregnant women. In the To inform optimal control strategies for anorectal STIs in women, first the prevalence of anorectal STI was determined by using routine universal collected data in the general STI clinic population of women. Such data is scarce, because of the general lack of a routine universal screening practice in women in STI control settings. Moreover, little is known about alternative transmission routes such as the anal use of fingers and/or sex toys. By assessing associations with medical and behavioural history, we aim to estimate the number of anorectal infections missed by the current practice of selective testing on indication and to formulate recommendations for control i.e., the testing and treatment strategies.The outpatient STI clinic of the South Limburg Public Health Service provides about 6000 consultations annually, offering free examination and treatment at three regional outpatient STI clinics. Between May 2012 and July 2013, three consultation nurses (out of 13) offered all their female patients aged 18 years and older (n = 663) routine testing for urogenital and anorectal chlamydia and gonorrhoea. This yielded a total of 654 consultations by 611 women for analysis (participation 92.2%). Fifty-two (7.8%) women declined an anorectal swab; reported reasons for non-participation were inconvenience (65%), fear (19%) and lack of necessity (16%). Non-participants were slightly younger than participants . Urogenital chlamydia prevalence was similar for non-participants and participants  versus 11.2% (73/654), P = 0.60). Gonorrhoea infections were not observed in both groups. The study was approved by the Medical Ethics Committee of Maastricht University (11-4-108).Chlamydia trachomatis and Neisseria gonorrhoeae using nucleic acid amplification assays according to the manufacturer’s procedure . Serum was tested for Treponema pallidum hemagglutination (TPHA) and HIV; all the women were TPHA and HIV negative. Each consult also included a standardised medical and sexual history taken by trained study nurses. It asked about self-reported symptoms and sexual behaviour in the past six months, i.e., ‘Did you practise anal sex in the past six months?’. Anal symptoms included rectal discharge, bleeding, pain, redness, burning sensation, or itching. Swingers were defined as women who were part of a male–female couple that had sex with other male–female couples and their self-identified heterosexual sex partners. Prostitutes were defined as women who reported having had sex for money in the past six months. Women who were prostitute and/or swinger were defined as prostitutes/swingers. All data was registered in an electronic patient registry.Women provided self-collected vaginal swabs and self-collected anorectal swabs, which studies have proven to be a generally acceptable, valid and feasible approach,24,25. TWomen were assigned to one of three non-overlapping hierarchically constructed indication categories based on reported behaviour and symptoms. Women in the “indication” category reported at least anal symptoms and/or anal sex, whether or not in combination with anal use of fingers and/or toys. Women who were assigned to the “fingers/toys” category only reported the anal use of fingers and/or toys and reported no anal symptoms and no anal sex. Women who reported no anal symptoms, no anal sex, and no anal use of fingers or toys were assigned to the “without indication” category. As no gonorrhoea infections were observed, statistical analyses focussed on chlamydia only. The prevalence of chlamydia was calculated by dividing the number of positive tests by the total number of tests, multiplied by 100. Univariate and multivariate logistic regression were used to identify determinants independently associated with anorectal chlamydia. Determinants tested were indication (with indication versus the two other categories combined), age categories, prostitutes/swingers (prostitutes and swingers versus other women), and use of fingers/toys (versus no use of fingers/toys). Anorectal infections in the categories “without indication” and “fingers/toys” were defined as infections missed by selective testing on indication as in current care. The share of infections missed was compared between indication categories, age categories (reference ≥ 29 years) and prostitutes/swingers using univariate and multivariate logistic regression. Interactions terms were added between indication categories, age categories and prostitutes/swingers in the multivariate models, but none were statistically significant and were removed from in the final models.Finally, to assess the anatomic site distributions of urogenital and anorectal chlamydia, all women who tested positive for chlamydia were assigned to a non-overlapping distribution category: (1) urogenital only, (2) urogenital and anorectal, or (3) anorectal only. Restricting to chlamydia positives, this variable was compared over indication categories (with indication versus the two other categories combined), age categories, and prostitutes/swingers using Fisher’s exact test. A P value of < 0.05 was considered statistically significant. Analyses were performed using SPSS version 17.0.0 . Written informed consent for participation in the study was obtained from participants. Written informed consent was not obtained from a parent or guardian.We included 654 consultations with an overall median participant age of 23 years (inter-quartile range: 21 to 34). The overall prevalence was 11.2% (73/654) for urogenital chlamydia and 8.4% (55/654) for anorectal chlamydia. Gonorrhoea infections were not observed. Overall, anal use of fingers was reported by 20.3% (133/654) of the women, anal use of toys by 8.9% (58/654), anal sex with a steady partner by 24.0% (157/654), anal sex with a casual partner by 13.1% (86/654) and anal symptoms by 3.1% (20/654). Anal symptoms reported were itching (n = 7), ulceration (n = 3), redness (n = 2), discharge (n = 1), pain/burning sensation (n = 5), bleeding (n = 3) and unspecified (n = 2). Only 3 women reported a combination of (two) symptoms.In total, 31.0% (203/654) of the women were assigned to the “indication” category , 7.3% (48/654) to the “fingers/toys” category , and 61.6% (403/654) to the “without indication” category . Women without indication were younger than women with indication (median of 22 years versus 36 years). The share of prostitutes/swingers was higher in women using fingers/toys only compared to women without indication  for women with indication and 8.6% (39/451) for the other women (categories without indication and fingers/toys) (P = 0.74). Prevalence in the three indication categories is displayed in Table 6/203 forIn total, 55 anorectal chlamydia infections were diagnosed; 67.3% (37/55) were diagnosed in women without indication Table . Only 2/Of all urogenital and anorectal chlamydia infections found, 68.4% (52/76) were concurrent urogenital and anorectal infections. Of all chlamydia infections, only three infections were isolated anorectal : two in women with indication and one in a woman without indication Figure . Of the This study revealed alarmingly high numbers of anorectal chlamydia in women. One in 12 women and even one in 7 young women was diagnosed with anorectal chlamydia. As over two thirds of these infections are currently being missed, current selective testing on indication of symptoms and anal sexual history is not an appropriate control strategy for anorectal chlamydia in women visiting an STI clinic. Almost all women with anorectal chlamydia had concurrent urogenital chlamydia (95%). To our knowledge, this is the first study with routine universal anorectal testing, i.e., independent of reported behaviour, symptoms or urogenital positivity, in a general group of women who visited an STI clinic and who took different sexual risks, including anal use of fingers and toys.Indication  was not associated with anorectal chlamydia. Yet current guidelines for anorectal STIs advocate selective symptom- and sexual history based testing for women,13. InclThe prevalence of anorectal chlamydia was substantial (8%) 55/654). Young age (≤ 21 years) was found to be associated with anorectal chlamydia showing a strikingly high 14% prevalence in this group of women, consistent with studies in high risk women visiting an STI clinic5/654. Yo. TherebyIn contrast to MSM, anorectal chlamydia in women was rarely isolated. In current study, one woman had an isolated anorectal infection but did not report anal sex or symptoms. Possible explanations for this could be underreporting, a false negative urogenital test,8, or auNevertheless, state of the art practice in chlamydia control entails the use of highly sensitive NAATs to test for chlamydia. Although NAATs are not yet FDA proved for anorectal testing, their use is highly recommended, accepted, and part of standard operating procedures in many care settings. A positWhen a more restricted policy is preferred, anorectal testing only in women with urogenital infection or direct treatment effective for both urogenital and non-urogenital chlamydia would detect and treat 95% of anorectal infections, since 52 of 55 anorectal infections had co-occurrence of urogenital chlamydia. However, for the former option delay between urogenital and anorectal tests and subsequent treatments could be a problem in practice. The substantial anorectal chlamydia prevalence and high co-occurrence with urogenital chlamydia fuels the need for debate on what is adequate treatment for anorectal chlamydia-21,23. TSeveral study limitations need to be acknowledged. We only included women who visited the STI clinic, so our results might not fully represent those that could be found in the general female population or within other healthcare settings. Although our instructions on specimen collection were clear, we cannot entirely rule out the possibility of specimen contamination . As women attending the STI clinic were randomly assigned to a consultation nurse, possible selection bias is likely minimal. Data on other high risk sexual behaviors  were not available, and their association with anorectal chlamydia in women could not be assessed. In our study, the prevalence or the proportion of infections missed by current selective testing in the non-participants is unknown. Eligible non-participants were slightly younger than participants. A study by Sethupathi et al. found women most at risk for anorectal infections included women aged < 20 years as was also found in current study. Therefore, the prevalence of anorectal infections may be underestimated in current study, yet due to the high response (93%), bias is expected to be minimal.In conclusion, prevalence of anorectal chlamydia in women was high and current selective testing on indication is not an appropriate control strategy to identify and treat anorectal chlamydia infections. Almost all women with anorectal chlamydia had concurrent urogenital chlamydia. More research is needed on the public health and clinical implications of anorectal chlamydia in women.http://www.icmje.org/coi_disclosure.pdf (available upon request from the corresponding author) and declare the following: no support from any organisation for the submitted work; no financial relationships with any organisations that might have an interest in the submitted work in the previous three years; no other relationships or activities that could appear to have influenced the submitted work.All authors have completed the ICMJE uniform disclosure form at CJPAH and NHTMDM were involved in the conception and design of the study. GAFSvL produced the database, analysed the data and wrote the first draft of the manuscript. All of the authors contributed to writing the paper; all had full access to all of the data in the study, and all can take responsibility for the integrity of the data and the accuracy of the data analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/274/prepub


Clostridium difficile infection (CDI) after taking an antibiotic (AB) and, at present, this opportunistic pathogen is one of the main causes of antibiotic-associated diarrhea in hospitalized patients. Here, we analyze the composition and functional differences in the gut microbiota of C. difficile infected (CDI) vs. non-infected patients, both patient groups having been treated with AB therapy. To do so we used 16S rRNA gene and metagenomic 454-based pyrosequencing approaches. Samples were taken before, during and after AB treatment and were checked for the presence of the pathogen. We performed different analyses and comparisons between infected (CD+) vs. non-infected (CD−) samples, allowing proposing putative candidate taxa and functions that might protect against C. difficile colonization. Most of these potentially protective taxa belonged to the Firmicutes phylum, mainly to the order Clostridiales, while some candidate protective functions were related to aromatic amino acid biosynthesis and stress response mechanisms. We also found that CDI patients showed, in general, lower diversity and richness than non-infected, as well as an overrepresentation of members of the families Bacteroidaceae, Enterococcaceae, Lactobacillaceae and Clostridium clusters XI and XIVa. Regarding metabolic functions, we detected higher abundance of genes involved in the transport and binding of carbohydrates, ions, and others compounds as a response to an antibiotic environment.Antibiotic therapy is a causative agent of severe disturbances in microbial communities. In healthy individuals, the gut microbiota prevents infection by harmful microorganisms through direct inhibition , competition, or stimulation of the host's immune defenses. However, widespread antibiotic use has resulted in short- and long-term shifts in the gut microbiota structure, leading to a loss in colonization resistance in some cases. Consequently, some patients develop  Clostridium difficile , preventing its growth and virulence, is still unknown. In this regard, direct antagonism was found in vitro since C. difficile is a target of bacteriocin produced by an intestinal strain of Bacillus thuringensis , metagenomics  and other meta-“omics”  have extended our knowledge of intestinal microbiota diversity and functions , considering C. difficile positive those samples that resulted positive for the three examined genes . Patients F and H were found positive after 16 and 35 days after AB treatment, respectively, whereas patient G was found positive on entering hospital , or presented diarrhea. The main features and therapy of all patients  are shown in Table In two previous studies we evaluated the effect of broad-spectrum antibiotics on five patients  through similar approaches of those presented in this work (16S rRNA gene and metagenomics) as part of the same research survey ] and centrifuged at 1250 g and 4°C for 2 min to remove fecal debris. The supernatants were centrifuged at maximum speed at 4°C for 5 min to pellet the cells. DNA was extracted with the QIAamp® DNA Stool Kit (Quiagen) following the manufacturer's instructions. Total DNA integrity was checked by running a standard agarose gel electrophoresis and the concentration was quantified with the QuantiT PicoGreen dsDNA Assay Kit (Invitrogen). For each sample, except of F_after from which there was no enough amount of DNA, the total DNA (metagenome) was directly pyrosequenced with a Roche GS FLX sequencer and Titanium chemistry in the Center for Public Health Research  . Thus, a total of 12 metagenomes were analyzed. We obtained a mean of 78,976 reads per sample with an average length of 374 bp.The fecal samples were resuspended in sterile PBS [containing, per liter, 8 g of NaCl, 0.2 g of KCl, 1.44 g of NaA region of the 16S rRNA gene  was amplified by polymerase chain reaction (PCR) for each sample. The primers were the universal E8F (5′-AGAGTTTGATCMTGGCTCAG-3′) with adaptor A and 530R (5′-CCGCGGCKGCTGGCAC-3′) with adaptor B using the sample-specific Multiplex Identifier (MID) for pyrosequencing according to 454 standard protocols. For each sample a 50 μl PCR mix was prepared, containing 5 μl of Buffer Taq (10X) with 20 mM MgCl2, 2 μl of dNTPs (10 mM), 1 μl of each primer (10 mM), 0.4 μl of Taq Fast start polymerase (5 u/μl), 39.6 μl of nuclease-free water and 1 μl of DNA template. PCR conditions were: 95°C for 2 min followed by 25 cycles of 95°C for 30 s, 52°C for 1 min and 72°C for 1 min and a final extension step at 72°C for 10 min. The amplification products were checked by electrophoresis in agarose gel (1.4%). PCR products were purified using NucleoFast® 96 PCR Clean-Up Kit (Macherey-Nagel) and quantified with the QuantiT PicoGreen dsDNA Assay Kit (Invitrogen). The pooled PCR products were directly pyrosequenced in the same way as the total DNA (described above). We obtained an average of 5394 reads per sample.We used the Ribosomal Database Project (RDP) pipeline   we calculated two diversity parameters: number of OTUs and Shannon index Shannon,  and two p-value with 10,000 bootstraps to estimate the probability of each cluster. This AU p-value indicates how strong the cluster is supported by data.We also performed a clustering based on OTU composition to study the similarity between samples using the pvclust library  and the Large Subunit rRNA Reference Database (LSUrdb) described in Urich et al.  was applied, implemented in the Vegan package under the R software. These approaches were applied to two different levels: the taxonomy based on the 16S rRNA gene, and the biological functions based on the TIGRFAMs annotations. We used the ShotgunFunctionalizeR package  and detrended correspondence (DCA) analyses were performed to explore the relationship between different groups of samples and with C. difficile colonization. Specifically, we performed three comparisons between groups of samples to identify taxa and functions that were significantly over-represented in CD− compared to CD+ samples. The taxa and functions resulting from the different comparisons were intersected to define the candidate protective group. For this purpose, we performed the following comparisons:We also used the “testGeneCategories.dircomp” test to identify taxa and metabolic functions that could play a protective role against p-value < 0.1) due to treatment, presumably allowing C. difficile overgrowth.Comparison 1.Since patients F and H were negative to the pathogen before treatment but positive during therapy, we compared the CD− samples before AB (F_before and H_before) against the CD positive samples (CD+) during AB   due to therapy or that changed less drastically than those in Comparison 1, since their presence could play a role in preventing C. difficile infection.Comparison 2. Since patients A, B, C, D and E did not develop CDI, we performed a comparison of the samples before AB treatment against their samples during treatment Table . We aimep-value < 0.05) could be incompatible with pathogen overgrowth as this was not detected.Comparison 3. Since patient H was negative for the pathogen 26 days after AB, we carried out a comparison of the CD+ samples of patient H (H35_D and H38_D) against the CD− sample taken after AB (H_after) Table . We aimeC. difficile colonization resistance.Finally, we intersected all these results to obtain a group of candidate taxa and functions that could participate in p-value below 0.01 were included in the network. Correlations and p-values were computed using the Spearman method implemented in R software.To find positive correlations between candidate protective taxa or functions found in the previous analyses and other taxa and functions obtained for samples from patients A, B, C, D and E during AB treatment , we performed Bayesian networks based on their relative abundance. The Bayesian networks were inferred using the bnlearn package Scutari,  in the RAll sequences have been entered in the European Bioinformatics Institute database, under accession numbers: ERP002192 , ERP001506 (patient E) and PRJEB5771 .Analysis of the gut microbiota of the three CDI patients  showed large variations in bacterial composition during therapy Figure .Akkermansia genus (30.6%) belonging to the family Verrucomicrobiaceae. Other bacterial families were also abundant such as Ruminococcaceae (20.8%), Oscillibacteriaceae  and Bacteroidaceae . When C. difficile was detected, at day 16 of AB treatment, all these taxa were almost absent in the community except Bacteroides, which had increased to 41.9%, becoming a predominant genus of the gut ecosystem. The Clostridium cluster XlVa increased dramatically (from 0.7% before AB course to 46.8% at day 16), being the most abundant group at this time point. After treatment, the abundance of the main taxa of the microbial community changed again, the predominant being Enterococcaceae , Streptococcaceae , Staphylococcaceae  and Clostridium cluster XI (3.5%).Before AB treatment, the bacterial composition of patient F was dominated by the C. difficile detection before, during, and after AB treatment, showing the most similar bacterial composition at the three time points, though there are some remarkable differences. The initial composition (G_before) consisted mostly of Bacteroidaceae  and Ruminococcaceae . During AB (G4_D), although Bacteroides decreased in abundance to 25%, it remained the most abundant genus, while Faecalibacterium (5.9%) decreased radically. However, Enterococcus increased during AB (from 1.3 to 14.9%). After therapy (G_after), Clostridium cluster XI became the predominant group (62.4%) whereas Streptococcus genus decreased progressively at each time point .Patient G was found positive to Escherichia genus, but its abundance decreased dramatically reaching the lowest values at days 35 and 38 of the broad-spectrum AB treatment , when C. difficile was detected. During days 7 and 14 of AB treatment the genus Bacteroides showed the higher abundance values (20.4 and 34.8%); however this taxon decreased on day 20, becoming undetectable by days 35 and 38. Streptococcus genus increased slightly in the two CD+ samples . The most striking shift occurred in the Lactobacillaceae family (Lactobacillus genus), whose frequency increased from less than 1% at the beginning of treatment to 83.3 and 70% on days 35 and 38 of the AB course, and was reduced to 15.5% after AB. We performed a statistical comparison to evaluate the differences in bacterial composition between the samples immediately prior to C. difficile detection  and in the CD+ samples .  by Enterobacteriaceae family, mainly ). Table . The maiThe three individuals exhibited great fluctuations in the number of observed OTUs, as well as in the diversity parameters analyzed Table . The divC. difficile presence contributed to explain the similarity pattern of the samples.Finally, we performed a cluster analysis to find similarities in microbiota composition between samples at OTU level (97%) Figure . The thrC. difficile infection , and thus all samples were CD−. To search for differences in microbiota composition possibly related to infection, we compared the 15 time points during the AB therapy of these CD− patients with samples from patients that were positive for C. difficile detection (CD+)   and the Chao1 estimator (p = 0.33). The gut microbiota of CD+ samples seems to be more heterogeneous and less rich than the CD− samples corresponding to patients that did not develop CDI, but a larger number of samples would be required to support this observation.First, we compared the Shannon index distributions between CD+ and CD− samples Figure . We founC. difficile infection is a factor that influences the microbiota structure. The factor proved to be significant with a p-value of 0.005.Thus, although the CD+ samples do not form a well-defined cluster, they share some features in their microbiota composition that differ from CD− samples.Second, we performed a detrended correspondence analysis (DCA) to explore the variations in bacterial composition between the same CD+ and CD− samples tested above Figure . The twoLactobacillus, Bacteroides, Enterococcus, Faecalibacterium, the family Lachnospiraceae incertae sedis, and the Clostridium clusters XIVa and XI, the latter included C. difficile. However, commensal members of the intestinal community, such as Roseburia, Coprococcus, Blautia, or Subdoligranulum genera and the families Erysipelotrichaceae and Ruminococcaceae were underrepresented . The three comparative analyses gave a number of statistically significant taxa , specifically to the families Ruminococcaceae , Oscillibacteraceae (Oscillibacter) and Eubacteriaceae (Anaerovorax). We also found unclassified Ruminococcaceae and Erysipelotrichaceae belonging to the Clostridiales and Erysipelotrichales orders, respectively, as well as other Clostridia and Clostridiales members. Finally, the genus Escherichia from the family Enterobacteriaceae, Proteobacteria phylum, was also detected.In order to obtain a subset of candidate bacteria that could be involved in xa Table , and intGemmiger, Subdoligranulum and uc_ Erysipelotrichaceae did not show any significant correlation and thus they are not represented in the figure. It is worth noting that most taxa showing a positive and significant correlation with the candidates were phylogenetically related to them, mainly belonging to the Clostridiales order, such as Roseburia and Coprococcus (Lachnospiraceae family) and Anaerotroncus (Ruminococcaceae family).Once the candidate protective taxa had been detected, we performed a Bayesian network  to find other related members of the bacterial community and hence also putatively involved in pathogen colonization resistance Figure . GemmigeIn the present work, we performed the functional annotation of the 12 metagenomes sequenced  by comparison against the TIGRFAM database, obtaining the following hierarchical classification: main roles , sub roles (more specific metabolic functions for each one of the main roles) and genes (metabolic functions) for all the reads. Figure In patient F, there were 51 significantly different sub roles between samples corresponding to before and during AB treatment (F_before vs. F16_D) Table . The mosC. difficile detection ; (ii) before vs. CD+ samples (H_before vs. H35_D and H38_D) and (iii) CD− prior to CDI vs. CD+ samples (H14_D and H20_D vs. H35_D and H38_D). In total, we found 37 significant sub roles increased or decreased in CD− samples. (i) Those that increased during AB but before CDI were mainly involved in “cell processes/DNA transformation” and “protein synthesis/translation factors,” whereas we observed a significant decrease in “amino acid biosynthesis/folic acid”; “mobile and extrachromosomal elements function/plasmid functions”; “signal transduction/PTS” and “transport and binding proteins/carbohydrates, organic alcohol and acids.” (ii). A similar functional profile was found when we compared before vs. CD+ samples. (iii) Finally, when we specifically compared the two samples previous to infection with the two CD+ samples we found 54 significant sub-roles. The most significant over-represented in the CD+ samples were: “signal transduction/PTS”; “transport and binding proteins/carbohydrates, organic alcohols, and acids”; “transport and binding proteins/amino acids, peptides and amines” and “cell envelope/biosynthesis and degradation of mureinsacculus and peptidoglycan” (Table Three different comparisons were made for patient H: (i) before vs. during treatment but before n” Table .p = 0) while “cell envelope/other” increased (p = 0.04).Patient G proved to be infected by the pathogen throughout the study. Thus, we compared the sub roles distribution, before vs. during treatment, to find those functions that could be AB-related. The comparison showed that only two categories changed during AB: “amino acid biosynthesis/serine family” decreased  those metabolic functions that may play a role in colonization resistance. Table We also performed a Bayesian network to find significant and positive associations between the candidate protective functions and other functions that may be important in pathogen infection resistance. Figure C. difficile (CD+ samples) after AB treatment and one patient (G) that despite not having taken AB was already CD+ when entered to the hospital. Patients F and H had an unusual microbiota at the start of the study (before AB treatment), enriched in Akkermansia genus (30.6%) and highly abundant in Escherichia genus (85.7%), respectively. We also compared the gut microbiota of those three patients with five individuals from two previous studies  that were positive for C. difficile, affect microbiota equilibrium  and a decrease in microbial diversity and species richness were found in individuals with CDI  can inhibit C. difficile growth  share some common features, being depleted in commensal genera such as Donskey, . EnterocAnaerovorax, Escherichia, Gemmiger, Oscillibacter, Ruminococcus, Subdoligranulum, uc_Clostridia, uc_Clostridiales, uc_Erysipelotrichaceae, and uc_Ruminococcaceae were found as candidates for protecting against C. difficile colonization. Bayesian correlation networks are a powerful tool to search and study ecological or metabolic associations in microbial communities  or recover from CDI (comparison 3), but decreased in those CD+ samples (Comparison 1). Thus, Roseburia or Coprococcus, are active anaerobic short-chain fatty acids (SCFA) producers (Barcenilla et al., C. difficile growth and also to decrease the production of toxin in vitro (May et al., C. difficile overgrowth (Bartlett, C. difficile colonization (Reeves et al., in vivo the colonization resistance capacity of the specific ensemble we have proposed.Some of the Clostridia members found to be associated to the main protective candidate taxa, such as C. difficile germination and growth. Related to this finding, a transcriptomic study revealed that sugars released by an altered microbiota are exploited by enteric pathogens such as Salmonella enterica and C. difficile (Ng et al., C. difficile and other opportunistic bacteria can efficiently catabolize the excess of carbohydrates generated by the disrupted microbiota and, in the absence of competitors, increase colonization rates.In a previous study, we showed that the metabolic profiles of AB-associated shifts in human gut microbiota were less dramatic than those in bacterial composition, principally when considering main roles. This is due to functional redundancy of the human gut microbiota, and the fact it has a very general set of functions (Pérez-Cobas et al., C. difficile colonization resistance (Table C. difficile. Also, some energy metabolism pathways seem important, such as TCA cycle, electron transport, or fatty acid biosynthesis. A great number of different transporter families, regulator genes, and genes involved in responses to osmotic or acid stress were also highlighted in the network, possibly playing a role in colonization resistance.Using the same three comparisons, we also found metabolic functions that may play a role in ce Table . Overallce Table . Future Another possible protective pathway was peptide catabolism via tryptophan metabolism. Low abundance of protein digestion markers was associated to susceptibility to CDI in the mouse gut (Theriot et al., Lactobacillus, Enterococcus, Clostridium clusters XIVa and XI but depleted in SCFA-producing bacteria. The latter bacterial group could be involved in C. difficile colonization resistance. A group of metabolic processes related to the metabolism of proteins, amino acids and responses to stress would seem to participate in avoiding pathogen invasion in the human gut ecosystem. Further research into these pathways should be undertaken to unravel the mechanism by which they participate in colonization resistance to C. difficile. A larger cohort of patients with similar sampling would be needed to deeper define the CDI microbiota at taxonomic and functional level.In summary, we found specific fecal microbiota in CDI patients as it was enriched in Andrés Moya, María J. Gosalbes, and Amparo Latorre conceived the work. Ana E. Pérez-Cobas performed all the analyses. María J. Gosalbes and Alejandro Artacho help with some of the analyses. The manuscript was written by Ana E. Pérez-Cobas, María J. Gosalbes, and Amparo Latorre. Andrés Moya and Stephan J. Ott revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Staphylococcus aureus is one of the main etiological agents of mastitis in ruminants. In the present retrospective study, we evaluated the potential interest of a previously described automated multiple loci Variable Number of Tandem Repeats (VNTR) Assay (MLVA) comprising 16 loci as a first line tool to investigate the population structure of S. aureus from mastitis. We determined the genetic diversity of S. aureus strains from cases of clinical and subclinical mastitis in dairy cattle , sheep (n = 18) and goats (n = 16). The 152 strains could be subdivided into 115 MLVA genotypes (including 14 genotypes for the ovine strains and 15 genotypes for the caprine strains). This corresponds to a discriminatory index (D) value of 0.9936. Comparison with published MLVA data obtained using the same protocol applied to strains from diverse human and animal origins revealed a low number (8.5%) of human-related MLVA genotypes among the present collection. Eighteen percent of the S. aureus mastitis collection belonged to clonal complexes apparently not associated with other pathological conditions. Some of them displayed a relatively low level of diversity in agreement with a restricted ecological niche. These findings provide arguments suggesting that specific S. aureus lineages particularly adapted to ruminant mammary glands have emerged and that MLVA is a convenient tool to provide a broad overview of the population, owing to the availability via internet of databases compiling published MLVA genotypes.The online version of this article (doi:10.1186/s13567-014-0097-4) contains supplementary material, which is available to authorized users. Staphylococcus aureus is one of the most prevalent etiologic agents of this disease in dairy cattle [Although several bacterial pathogens can cause mastitis, y cattle , and they cattle ,3. As anS. aureus mastitis in dairy cattle has been studied using various molecular typing methods. Techniques that rely on the comparison of electrophoretic patterns, such as Pulsed-Field Gel Electrophoresis (PFGE) [spa typing overcome these problems by producing sharable and easily storable numeric-format results [spa typing is based on sequencing of a highly polymorphic tandem repeat locus showing internal variations of repeat units. When applied to S. aureus, MLST has a low discriminatory power for a relatively high cost, so that some investigators are evaluating the practical feasibility of replacing MLST by whole genome sequence analysis [spa locus typing alone is not always a robust indicator of genetic background, as illustrated for instance in an ST398 investigation [S. aureus subtyping [spa. In addition to its much higher discriminatory power as compared to previous techniques, MLVA was able to correctly predict clonal complex (CC) assignment and consequently benefit from the strong phylogenetic content provided by MLST analysis [S. aureus genotyping using 8 or 16 VNTR loci have been published [Orsay was demonstrated as being highly suitable for genotyping S. aureus isolates from human, animal and food sources [During the past decade, the epidemiology of s (PFGE) -6, Randos (PFGE) , ribotyps (PFGE) ,8 and Mus (PFGE)  proved t results -13. MLSTanalysis ,15. spa tigation  and is otigation . During ubtyping -20 and ranalysis . Automatublished ,22. The S. aureus strains. These studies described CC including S. aureus mainly isolated from humans  or animals  [In recent years several MLST-based studies investigating mastitis ,13,23 had CC705) ,24-27. RS. aureus strains from mastitis in dairy cows, goats and sheep from different countries and regions. Through the additional information provided by the analysis of VNTR allele distribution, the objective was also to better identify the evolution and emergence of host-adapted or udder-adapted clones.The aim of the present study was to use MLVA for the first time to infer a population structure of n = 118), ovine (n = 18) and caprine (n = 16) clinical or subclinical mastitis. An additional table file shows this in more detail  with lysostaphin . Nucleic acid quality and concentration were analysed using an ND-1000 spectrophotometer . Diluted samples of 5 ng/μL in distilled water  were used as DNA template for PCR amplification.Orsay were amplified in two multiplex PCR using the CeeramTools® Staphylococcus typing kit  as previously described [Orsay data derived from the 152 strains of this study were compared to published data obtained with the same method [S. aureus strains isolated from human (n = 106), swine (n = 32), poultry (n = 30), companion animals (n = 17), horse (n = 5), small ruminant (n = 11), rodent (n = 2), cattle (n = 1), food (n = 34) and food poisoning events (n = 13) were characterized by MLVA16Orsay complemented by MLST and spa typing. This reference dataset is now used to link new strains. All unclustered strains from the present investigation were characterized by MLST and spa typing.The 16 VNTR loci included in MLVA16escribed . The typescribed . Simpsonescribed . The MLVe method  in ordere method ,22,30. Ispa tandem repeat amplification and MLST analysis were as previously described [spa repeat nomenclature was that of Shopsin et al. [spa types were retrieved from [The primers and condition used for the escribed ,31,32. Tn et al.  and spa ved from . MLST alved from .t-tests. Differences were considered significant when p < 0.05.Data were analysed by chi-square or Student’s spa typing and MLST analysis of selected strains. A dendrogram deduced from the clustering of the 152 S. aureus mastitis-associated strains is presented in Figure spa types. CC8 and CC30, frequently associated with human S. aureus infections, and CC425 a common ovine genotype  and goat strains (collected from six herds in 2004) were segregated by MLVA typing. These ten strains came from three different French husbandry regions without any contact with each other. CC1, CC8, CC9, CC20, CC97, CC151, CC398 and CC479 included only bovine strains. CC133 was the sole cluster showing complete host diversity: 13 bovine strains originating from different collection sites (11% of the bovine isolates), eleven sheep strains (61% of the ovine isolates) and eight goat strains (50% of the caprine strains) belonged to this CC. The remaining small ruminant strains were clustered in CC30, CC425 or were singletons. Only one MLVA genotype comprised strains of two different host species: the cow and goat . No MLVA genotype was common to goat and sheep.Figure p < 0.001).Bovine isolates from the three countries fell into 6 up to 10 different CC; small ruminant isolates fell into 3 or 4 CC. The CC distribution was different between Germany, France and Brazil . The diversity indexes were lower in the latter than in the human-related (p < 0.05) or animal-specific (NS) clusters. An additional table file shows this in more detail  and Sa0387 (p < 0.0001). An additional file shows this in more detail  from six different lineages  might be of human origin. Among these lineages, CC30 has also been shown to be established in swine . Devries species . Several% from sied hosts ,9,37. Coed hosts . CC20 sted hosts ,40 and ied hosts  but are ed hosts ,42,43.Almost half of the collection studied belonged to two major animal-predominant CC (CC97 and CC133). Twenty-nine and 26 MLVA16 genotypes were observed for a total of 34 CC97 strains and 32 CC133 strains respectively. CC97 is a widespread bovine lineage largely responsible for bovine mastitis cases in Chile, Brazil, Japan and the United States , and alsIn the present study, we identified three additional clusters putatively showing strong association with the mammary gland tissue: the “bovine” CC151 (eleven strains) and CC479 (six strains), and the “small ruminants” CC130 (ten strains). All together, they represent 18% of the present collection. These lineages have almost never been isolated from humans and, moreover, were almost exclusively sampled from intramammary infections ,37,49-51S. aureus carriage is very limited as compared to humans.From the population structure point of view, one must remain careful when interpreting the observation of a small number of CC comprising the majority of livestock and especially mammary isolates. Recently, a single publication identified 9 CC and 4 ST in ovine and caprine mastitis , whereasMycobacterium tuberculosis, [S. aureus. Indeed in a preliminary study, we observed that at least some tandem repeats may have an effect on the transcription level of adjacent genes indicating that they are not neutral. Within the same CC, the transcription level of the gene located immediately downstream from Sa0906 was five-fold higher in a strain with four repeat units as compared to a strain with one repeat unit . Intereculosis, ,56. Becaculosis, , whole gblished; ).versus environmental herd mastitis) is a critical point to implement relevant control measures. In the case of S. aureus, generally classified as a contagious pathogen, the molecular epidemiology profile is in some herds of the environmental type [The characterization of mastitis transmission models (contagious tal type . Taking tal type  demonstrtal type ,62.


Progranulin (PGRN), a pleiotropic growth factor, has emerged as an immunoregulatory molecule. Because the roles of PGRN in dermatomyositis (DM) are still unknown, we investigated whether serum PGRN levels are associated with disease activity and prognosis in DM patients, particularly in those with DM complicated with interstitial lung disease (ILD).The serum levels of PGRN were measured by enzyme-linked immunosorbent assay in patients with DM : n =17, chronic interstitial pneumonia (CIP): n =24, without ILD: n =16), polymyositis  and normal healthy controls . We assessed the correlation between the serum PGRN levels and the activity indexes of ILD or prognosis in DM patients with ILD.P =0.0025) and in NHCs (P <0.0001). In DM patients, the levels were significantly higher in patients with A/SIP than in those with CIP (P <0.0001) or without ILD (P =0.0003). The serum PGRN levels in DM patients with ILD significantly correlated with serum ferritin , lactate dehydrogenase  and C-reactive protein  levels. Moreover, in DM patients with ILD, the cumulative survival rate for 6 months was significantly lower in the group with serum PGRN levels ≥200 ng/ml (67%) than in the group with serum PGRN levels <200 ng/ml (100%) (P =0.0009).Serum PGRN levels were significantly higher in DM patients than in PM patients (Serum PGRN is associated with disease activity and prognosis of DM with ILD. PGRN may play a role in the pathogenesis of DM and could be a useful biomarker. Dermatomyositis (DM) is a systemic autoimmune and inflammatory disease that involves not only the muscle and skin but also several other organs, such as the lungs, heart and joints . InterstProgranulin (PGRN) [GenBank:NC_000017] is an extracellular glycoprotein containing seven and one-half repeats of cysteine-rich motifs. PGRN is proteolytically cleaved by extracellular proteases, such as proteinase 3 (PR3) and elastase, into granulin (GRN)  that ranHere, we show that serum PGRN levels are significantly elevated in DM patients, particularly those with A/SIP, and are associated with disease activity and prognosis of DM patients with ILD. PGRN may have a role in the pathogenesis of DM.We performed a cross-sectional study of patients who were treated for DM or polymyositis (PM) at Kyushu University Hospital, Saga University Hospital and Japanese Red Cross Fukuoka Hospital between 2002 and 2013. Fifty-seven Japanese patients with DM and twenty-one patients with PM were enrolled, and sera were obtained from these patients. All of the patients fulfilled the criteria of Bohan and Peter  for DM oThe serum PGRN levels were determined using enzyme-linked immunosorbent assay (ELISA) kits  according to the manufacturer’s protocol. Briefly, calibrators, control sera and patients’ sera (stored samples) were diluted and incubated with a mouse monoclonal antibody against PGRN adsorbed onto the microtiter plate wells. After washing, a mouse monoclonal antibody against PGRN conjugated to horseradish peroxidase was added, followed by a second washing step and the addition of tetramethylbenzidine substrate. The intensity of the blue color developed was in proportion to the amount of PGRN bound in the initial step. The reaction was terminated by the addition of 2 N sulfuric acid. The absorbance was measured in a microtiter plate reader  and converted into nanograms per milliliter by plotting the values against the PGRN titer of the calibrators/standards given by the manufacturer. The assay range was 1.56 to 100 ng/ml.ILD was assessed by radiography and high-resolution computed tomography of the chest. ILD with DM was divided into two forms: A/SIP and CIP. A/SIP is defined as a rapidly progressive ILD within 3 months from the onset of symptoms and radiographically diffuse alveolar damage pattern or nonspecific interstitial pneumonias (NSIP). CIP is defined as an asymptomatic, non–rapidly progressive ILD or slowly progressive ILD over the course of 3 months and radiographically confirmed NSIP or bronchiolitis obliterans with organizing pneumonia pattern, according to the guidelines of the International Consensus Statement of idiopathic Pulmonary Fibrosis of the American Thoracic Society and the European Respiratory Society . The desU test. Fisher’s exact test was used for comparison of frequencies. The Steel-Dwass test was used for multiple comparisons among all groups. The relationships between PGRN levels and other continuous variables were analyzed using Spearman’s rank correlation. Receiver operating characteristic (ROC) analyses were performed for a mathematical expression of different serum PGRN levels as cutoff points. The cumulative survival rate was calculated using the Kaplan-Meier test. The log-rank test was also used to compare survival. P-values less than 0.05 were considered significant. All tests were two-tailed. All analyses were performed using JMP statistical software .The differences between two groups were analyzed using the Mann–Whitney Of the 57 patients with DM enrolled in the present study, 49 were women, and 8 were men . These patients ranged in age from 30 to 87 years (median age: 53 years). Of the 21 patients with PM, 16 were women, and 5 were men . These patients ranged in age from 15 to 85 years (median age: 53 years). Among the NHCs, 51 were women, and 9 were men. They ranged in age from 20 to 59 years (median age: 32 years). There were significant differences between patients with DM or PM and NHCs in terms of age. However, there was not a significant correlation between serum PGRN levels and age in NHCs.P <0.0001). Strikingly, serum PGRN levels in patients with DM (median: 109 ng/ml) were significantly and markedly higher than in PM patients (P =0.0025) and in healthy controls (P <0.0001).To investigate the association of PGRN with DM, we first compared serum PGRN levels among 57 patients with DM, 21 patients with PM and 60 NHCs by using ELISA  or without ILD . There was no significant difference between serum PGRN levels in DM patients with CIP and those without ILD (P =0.8382). However, serum PGRN levels in DM patients with CIP or without ILD were significantly higher than in NHCs .Because DM is occasionally complicated with ILD -10, we nTo evaluate clinical manifestations other than serum PGRN levels, we compared those between DM patients with A/SIP and without A/SIP Table . There wrS =0.54, P =0.0003), ferritin  and CRP . However, there was not a significant correlation between serum levels of PGRN and KL-6 . In five DM patients with active ILD that had subsided with treatment, serum PGRN levels decreased in all of them   Table . ImmunosP =0.0009). These results suggest that baseline serum PGRN levels predict the prognosis of DM patients with ILD.Because DM with ILD is occasionally life-threatening ,5,7, in To our knowledge, this is the first study to show that PGRN levels were significantly elevated in sera of DM patients, in particular those with A/SIP, and these concentrations were associated with the disease activity and prognosis of DM patients with ILD.Consistent with previous reports ,12, the In the present study, other than DM, we also measured concentrations of serum PGRN in patients with PM (n =21), including 6 with ILD (median: 59 ng/ml) and 15 with systemic sclerosis (median: 49 ng/ml) (same levels in NHCs), including 13 with ILD (median: 49 ng/ml). In our data, although sera from PM patients contained significantly elevated PGRN compared with NHCs, DM patients’ sera had much higher levels of PGRN, especially among DM patients who also had A/SIP. This difference may occur because activated macrophages appeared especially in DM patients with A/SIP. Although we could not recruit acute respiratory distress syndrome (ARDS) patients into this study, ARDS could be an additional control to determine whether PGRN is a more general marker of acute respiratory inflammation.PGRN is reported to be important in the initiation of inflammation by recruiting fibroblasts, macrophages and neutrophils to the site of inflammation . PGRN isrS =0.54, P =0.0003) . However, there was no significant correlation between serum levels of PGRN and those of KL-6, another biomarker for ILD. Recent studies suggest serum ferritin levels as a marker for severity of acute progressive ILD in DM and CADM patients [Serum PGRN levels showed a significantly positive correlation with serum levels of LDH (3) Table  and ferrpatients -14. The patients . LDH andpatients . KL-6 rapatients . Thus, PrS =0.48, P =0.0015)  than in those with CIP (37.5%) and in those without ILD (5.9%). CADM may be a useful indicator for the occurrence of A/SIP. The levels of CRP showed significant differences between DM patients with A/SIP and those without and correlated with serum PGRN levels (5) Table . We prevMost of the DM patients with CIP were treated with PSL and immunosuppressive agents. HSCT was performed for one DM patient with CIP and a refractory skin ulcer. In contrast, several patients with A/SIP required combination therapy with PSL, CNI and IVCY (and HSCTs). This distinction might be responsible for cellular phenotypes affecting the pathogenesis of ILD.P =0.0009) (Figure The cumulative survival rate was significantly lower in the group with serum PGRN levels ≥200 ng/ml than in the group with serum PGRN levels <200 ng/ml  Figure . These dWe must acknowledge some limitations of this study. The sample size of our study was small. Statistical tests usually require a larger sample size to justify that the effect did not happen by chance alone. Moreover, owing to the study’s cross-sectional design, it is difficult to establish the exact and definite causal relationships, except the association between PGRN and development of ILD with DM, on the basis of the collected data. There were potential confounding effects of medication use on serum PGRN levels in patients with inactive DM with CIP and without ILD. Last, to fully validate PGRN as a predictive biomarker, we need to show that elevated PGRN levels predate clinical respiratory decompensation.In conclusion, this pilot study demonstrates that the serum PGRN levels were elevated in patients with DM, in particular those with A/SIP, and were correlated with disease activity and prognosis of DM patients with ILD. PGRN could be a useful biomarker for disease activity and a predictor of survival in DM patients with ILD. These findings of the study will provide new insights into the pathogenesis as well as the therapy of DM, and will shed new light on the dysregulation of the immune system in autoimmune diseases. Further studies are required to reveal more precisely the mechanisms of PGRN in human autoimmune diseases.


Aims. Sleep disorders have recently become a significant public health problem worldwide and have deleterious health consequences. Obstructive sleep apnea (OSA) is the most common type of sleep-related breathing disorders. We aimed to evaluate anthropometric measurements, glucose metabolism, and cortisol levels in patients with obstructive sleep apnea (OSA). Materials and Methods. A total of 50 patients with a body mass index ≥30 and major OSA symptoms were included in this study. Anthropometric measurements of the patients were recorded and blood samples were drawn for laboratory analysis. A 24-hour urine sample was also collected from each subject for measurement of 24-hour cortisol excretion. Patients were divided equally into 2 groups according to polysomnography results: control group with an apnea-hypopnea index (AHI) <5 (n = 25) and OSA group with an AHI ≥5 (n = 25). Results. Neck and waist circumference, fasting plasma glucose, HbA1c, late-night serum cortisol, morning serum cortisol after 1 mg dexamethasone suppression test, and 24-hour urinary cortisol levels were significantly higher in OSA patients compared to control subjects. Newly diagnosed DM was more frequent in patients with OSA than control subjects . There was a significant positive correlation between AHI and neck circumference, glucose, and late-night serum cortisol. Conclusions. Our study indicates that increased waist and neck circumferences constitute a risk for OSA regardless of obesity status. In addition, OSA has adverse effects on endocrine function and glucose metabolism. Obstructive sleep apnea (OSA) is the most common type of sleep-related breathing disorders. This disorder is characterized by excessive daytime sleepiness, snoring, and episodes of apnea. Cardiovascular diseases, which are among long-term adverse effects of OSA, lead to increased mortality and morbidity . The onlIn order to sustain life, the hypothalamus-pituitary-adrenal (HPA) axis should function to elicit the normal cortisol response to stress. In stress-free states the normal cortisol release shows a diurnal pattern; that is, the highest cortisol levels are observed early in the morning (04:00–08:00), while the lowest levels occur late at night (02:00–04:00). Patients with OSA frequently exhibit increased ventilatory effort, sleep fragmentation, and oxygen desaturation. These events produce stress in an individual's body, activating the HPA axis that increases cortisol production. Cortisol has many functions including regulation of blood glucose, vascular tone, and myocardial contractility.OSA increases the risk of hypertension, diabetes mellitus (DM), myocardial infarction, congestive heart failure, and stroke . In our A total of 50 patients with a body mass index ≥30 and major OSA symptoms including snoring, daytime sleepiness, and witnessed apnea were included in this study. We conducted this study between January 2012 and September 2013 at Department of Endocrinology, Faculty of Medicine, Dicle University. We obtained approval from a local medical research ethics committee. All the following types of patients were excluded from our study: patients with previous diagnosis of OSA, continuous positive airway pressure (CPAP) treatment, or bilevel positive airway pressure (BiPAP) therapy, patients with type 2 DM, hypertension, hypothyroidism, Cushing syndrome, hyperaldosteronism, pregnancy, malignancy, congestive heart failure, psychotic disorder, chronic liver failure, nephrotic syndrome, or chronic renal failure, or patients using drugs that had a potential to impair study tests . All subjects entering the study gave a written informed consent prior to the study, and all study tests and examinations were performed in compliance with the Helsinki Declaration regulating biomedical research on humans.Blood samples for fasting glucose, insulin, HbA1c, adrenocorticotropic hormone (ACTH), and cortisol were drawn at 08:00 from each patient during their stay in the endocrinology department. Newly diagnosed diabetes mellitus was determined according to ADA criteria . Homeostμg/dL (50 nmol/L) suggested a suppressed cortisol level. A low dose dexamethasone suppression test with 2 mg dexamethasone was performed for 2 days in patients with high cortisol levels.An additional blood sample was drawn for cortisol level at 23:00. A 1 mg dexamethasone suppression test was performed on the next day. A 1 mg decort tablet was administered at 23:00 and cortisol level was measured at 08:00 the next morning. A cortisol level less than 1.8 2 (kg/m2). Waist circumference was measured from the diameter between the costal arch and the anterior superior iliac spine at umbilicus level. Neck circumference was measured from the level of superior border of cricothyroid membrane. The same person performed all the measurements using the same tools; the results were rounded to the closest 0.5 cm to minimize measurement errors.Body weight, length, waist circumference, and neck circumference were measured with a nonflexible tape and recorded in centimeters. Body mass index was calculated with the formula of body weight/heightAfter completion of the procedures, each patient had a polysomnographic recording at Dicle University, Department of Chest Diseases, Center of Sleep Disorders. The polysomnographic recordings were carried out using a 32-channel E series polysomnography device . The patients were monitored for at least 8 hours during sleep. Electroencephalography (EEG) recordings were made from 4 channels designated as C3/A2, C4/A1, O1/A2, O2/A1 according to the International 10–20 system that determines the locations of the electrodes. Left-right electrooculography (EOG), mental electromyography (EMG), and electrocardiography (ECG) recordings were also performed.2) was measured with a pulse oximetry during sleep. Leg movements were recorded with a bilateral pretibial EMG. PSG recordings were scored according to the Rechtschaffen and Kales criteria as 30-second epochs. All recordings were scored manually in a digital format by a physician certified in sleep disordered breathing. Sleep stages, respiratory events, and their characteristics were recorded. Hypopnea was defined as a reduction in nasal pressure signal of ≥50% that lasted ≥10 s, resulting in a ≥3% decrease in oxygen saturation from the prevent baseline or an arousal.A transducer system connected to a nasal cannula was used to record respiratory events. The respiratory movements were recorded with the help of thoracic and abdominal tapes. Snoring was monitored with a microphone placed on the larynx at the anterosuperior portion of the neck. Oxygen saturation , Version 18.0 for Windows . Normally distributed variables were presented using means and standard deviations. Student'sn = 25) and OSA group (n = 25). The mean age was 43.76 ± 8.36 year and the male-to-female ratio was 13 : 12 in the control group. In the OSA group, the mean age was 46.68 ± 5.75 years with a male-to-female ratio of 12 : 13. There were no differences between groups for age and gender distribution (p > 0.05).A total of 50 patients were enrolled in the study and patients were divided equally into 2 groups according to polysomnography results: control group (p < 0.01) and waist circumference  were significantly higher in OSA group compared to control group. Body mass index was not significantly different between the groups . Demographic characteristics and anthropometric measurements of the groups are presented in r = 0.477, p < 0.001; When comparing anthropometric measurements between the control group and OSA group, neck circumference  and HbA1c . There was no significant difference between groups in insulin levels and homeostatic model assessment insulin resistance (HOMA-IR). Newly diagnosed DM was more frequent in patients with OSA than control subjects . Results are shown in r = 0.480, p < 0.01; Compared to control subjects, OSA patients had significantly higher levels of fasting plasma glucose , morning serum cortisol after 1 mg dexamethasone suppression test  and 24-hour urinary cortisol  levels were significantly higher in the OSA group than control group. However, morning serum cortisol and adrenocorticotropic hormone (ACTH) levels were not significantly different between the groups. There was a significant positive correlation between AHI and late-night serum cortisol . When a 2 mg dexamethasone suppression test was applied, plasma cortisol levels were suppressed in both group.A 1 mg dexamethasone suppression test was administered to all patients. Plasma cortisol levels were not suppressed in 1 of 25 patients (4%) in the control group and in 5 out of 25 patients (20%) in the OSA group . This finding supported the idea that neck circumference alone is a risk factor for OSA independent of obesity.Therefore, it is possible that obesity increases susceptibility to OSA. The risk for OSA increases 8–12 times in persons with a BMI greater than 28 . This riRepetitive respiratory difficulties during sleep cause intermittent hypoxia and alterations in intrathoracic pressure that trigger autonomic responses. Among these autonomic alterations, increased sympathetic activity plays an important role. Increased sympathetic activity, sleep fragmentation, and intermittent hypoxia contribute to metabolic dysfunction , 15. Spep = 0.034). As patients with OSA are usually obese, their risk of having or developing insulin resistance, type 2 DM, and hypertension is increased. Nocturnal cortisol elevations in these patients further increase the already heightened risk. The prevalence of type 2 DM is around 30% in patients with OSA [In a 270-patient study, Ip et al.  grouped with OSA .2/day [Cortisol release shows a diurnal variation in normal people. The HPA axis secretes cortisol in a pulsatile manner at morning hours and sets cortisol to release into blood stream at a minimum level at night. That is, the highest cortisol levels are observed early in the morning (04:00–08:00) while the lowest ones occur late at night (02:00–04:00). Daily cortisol production is at a level of 5.7 mg/m2/day . An esti2/day . In stre2/day , 22. Sle2/day . Sympathp < 0.05). The severity of OSA worsens as the AHI increases. Thus, frequent awakenings and duration of hypoxia also increase. Also in our patients an increase in AHI was correlated with a nocturnal cortisol elevation , suggesting that stress and night wakefulness boost the activity of HPA axis and increase pulsatile cortisol release.Various studies found no difference between OSA patients and the control group with respect to morning cortisol levels, although nocturnal cortisol levels have been found to be higher in OSA patients –26. Grunp < 0.01). This suggests that it is more difficult for OSA patients to suppress cortisol with a 1 mg dexamethasone suppression test. Patients with OSA had a higher 24-hour urinary cortisol level compared to the control group. These two findings support nocturnal cortisol elevation in patients with OSA which is in line with the two previous studies [All of our patients underwent a 1 mg dexamethasone suppression test. The cortisol level was 0.87 ± 0.45 mcg/dL in the control group and 1.53 ± 1.09 mcg/dL in the OSA group ( studies .Sleep disorders have recently become a significant public health problem both in our country and worldwide. As the number of people with obesity and obesity-related diseases increases, the number of patients suffering from OSA increases. Obesity, along with increased neck and waist circumferences, increases the risk of OSA. Hypoxemia and stress induced by OSA further worsen the impact of obesity on metabolic and endocrine systems.This study reveals that recognition of newly diagnosed type 2 diabetes mellitus in 32% of OSA patients and especially increased nocturnal cortisol levels in OSA patients both point to an adverse effect of OSA on the endocrine system. One additional significant finding of our study was that increased waist and neck circumferences constituted a risk for OSA regardless of obesity status. We suggest that endocrinological and anthropometric examinations are necessary for OSAS patients. We hope that the current study will contribute significantly to the literature. A limitation of the study was the relatively small sample size; therefore further division of OSAS group could not be examined. We also believe that further studies with larger sample size are needed on this subject.