Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is characterized by unexplained persistent fatigue, commonly accompanied by cognitive dysfunction, sleeping disturbances, orthostatic intolerance, fever, lymphadenopathy, and irritable bowel syndrome (IBS). The extent to which the gastrointestinal microbiome and peripheral inflammation are associated with ME/CFS remains unclear. We pursued rigorous clinical characterization, fecal bacterial metagenomics, and plasma immune molecule analyses in 50 ME/CFS patients and 50 healthy controls frequency-matched for age, sex, race/ethnicity, geographic site, and season of sampling.Alistipes and decreased Faecalibacterium emerged as the top biomarkers of ME/CFS with IBS; while increased unclassified Bacteroides abundance and decreased Bacteroides vulgatus were the top biomarkers of ME/CFS without IBS. Despite findings of differences in bacterial taxa and metabolic pathways defining ME/CFS subgroups, decreased metabolic pathways associated with unsaturated fatty acid biosynthesis and increased atrazine degradation pathways were independent of IBS co-morbidity. Increased vitamin B6 biosynthesis/salvage and pyrimidine ribonucleoside degradation were the top metabolic pathways in ME/CFS without IBS as well as in the total ME/CFS cohort. In ME/CFS subgroups, symptom severity measures including pain, fatigue, and reduced motivation were correlated with the abundance of distinct bacterial taxa and metabolic pathways.Topological analysis revealed associations between IBS co-morbidity, body mass index, fecal bacterial composition, and bacterial metabolic pathways but not plasma immune molecules. IBS co-morbidity was the strongest driving factor in the separation of topological networks based on bacterial profiles and metabolic pathways. Predictive selection models based on bacterial profiles supported findings from topological analyses indicating that ME/CFS subgroups, defined by IBS status, could be distinguished from control subjects with high predictive accuracy. Bacterial taxa predictive of ME/CFS patients with IBS were distinct from taxa associated with ME/CFS patients without IBS. Increased abundance of unclassified Independent of IBS, ME/CFS is associated with dysbiosis and distinct bacterial metabolic disturbances that may influence disease severity. However, our findings indicate that dysbiotic features that are uniquely ME/CFS-associated may be masked by disturbances arising from the high prevalence of IBS co-morbidity in ME/CFS. These insights may enable more accurate diagnosis and lead to insights that inform the development of specific therapeutic strategies in ME/CFS subgroups.The online version of this article (doi:10.1186/s40168-017-0261-y) contains supplementary material, which is available to authorized users. Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is an unexplained, chronic debilitating disorder that in the USA alone is estimated to affect up to 2.5 million people, with annual costs of $24 billion , 2. DiagBacteria, their metabolites, and the host molecules they influence are participants in bidirectional communication pathways linking the gut and the central nervous system (CNS) , 8. InteCulture-based and 16S ribosomal RNA (rRNA) gene sequencing studies of stool bacteria have revealed evidence of dysbiosis  in ME/CFS –14. Alte2).Subjects included 50 ME/CFS cases and 50 healthy controls recruited at four sites across the USA  who met the 1994 CDC Fukuda  and the Shotgun metagenomic sequencing of fecal samples was pursued to determine microbial composition (relative abundance of taxa) and infer bacterial metabolic pathways in the ME/CFS and control subjects. An average of 7 Gb of sequence per sample  was generated using high-throughput sequencing. Levels of plasma immune molecules were quantitated by immunoassay. We built a TDA network comprised of 100 samples (50 cases and 50 controls) and 1358 total variables. The variables consisted of the following elements: 574 representing the relative abundance of bacterial taxa; 586 representing metabolic pathways ; 61 reflecting levels of each plasma immune molecule in the assay; 80 representing symptoms ; and 57 co-morbidities and demographic variables.Relationships among these datasets were analyzed using TDA (AYASDI software) to identify multidimensional networks and the individual factors  that distinguish those networks.Lachnospiraceae and Porphyromonadaceae were lower in the ME/CFS (both with and without IBS) compared to the controls, whereas the relative abundance of the family Clostridiaceae was higher. At the genus level, the abundances of Dorea, Faecalibacterium, Coprococcus, Roseburia, and Odoribacter were lower in the ME/CFS compared to the controls, whereas abundances of Clostridium and Coprobacillus were higher. The 12 bacterial species driving the differences between the ME/CFS and control groups were Faecalibacterium prausnitzii, Faecalibacterium cf., Roseburia inulinivorans, Dorea longicatena, Dorea formicigenerans, Coprococcus catus, Odoribacter splanchnicus, Ruminococcus obeum, and Parabacteroides merdae  and Clostridium asparagiforme, Clostridium symbiosum, and Coprobacillus bacterium .The ME/CFS subjects formed separate topological networks from the control subjects in TDA Fig. . IBS co-Proteobacteria phylum, C. catus, and F. prausnitzii species and increased representation from the Clostridiaceae family compared to the controls. Superpathway analysis showed alteration in heme biosynthesis, carboxylates, amino acid (AA), and polyamine metabolism. In the ME/CFS without the IBS group, the difference was driven by the increased abundance of members of the Clostridiaceae family, the Clostridium, and Pseudoflavonifractor genera, and the decreased abundance of members of the Porphyromonadaceae family and Odoribacter and Parabacteroides genera. The bacterial species driving the differences between the ME/CFS without IBS and control groups were D. formicigenerans, C. catus, Blautia hansenii, and Parabacteroides distasonis  and unclassified Bacteroides, D. longicatena, Ruminococcus gnavus, C. symbiosum, Eggerthella lenta, Pseudoflavonifractor capillosus, C. bacterium, Clostridium cf. and scindens . The ME/CFS subgroup comparison between the ME/CFS + IBS and ME/CFS without IBS is shown in Additional file Intragroup variability in the control group was smaller than in the ME/CFS group Fig. . The TDAFour networks defined by IBS co-morbidity and BMI  and Firmicutes   were represented at low relative abundance (mean relative abundance <5%) in samples.Compositional taxonomic analysis based on metagenomic sequencing indicated that the two dominant phyla in both ME/CFS and control individuals were ly) Fig. . Combinep < 0.001; PC2 10.69%, p = 0.03). Within control subjects, BC dissimilarity was significantly lower than within ME/CFS subjects, consistent with our findings based on TDA analysis  but was not higher than the within ME/CFS comparisons  based on the species-level Bray-Curtis  dissimilsis Fig. . The betons Fig. . TogetheU test with Benjamini-Hochberg correction (p < 0.05 and adjusted p < 0.2), 41 bacterial species, genera, families, or orders differed between the ME/CFS and control groups  and unclassified Bacteroides, unclassified Alistipes, P. capillosus, Clostridium bolteae, R. gnavus, C. asparagiforme, Anaerostipes caccae, C. bacterium, C. symbiosum, and C. scindens . Species in Faecalibacterium, Roseburia, Dorea, Coprococcus, Clostridium, Ruminococcus, and Coprobacillus species were significantly different in the ME/CFS patients compared to the controls by TDA, LEfSe, and nonparametric testing ) identified 22 bacterial taxa enriched in ME/CFS and 27 enriched in controls Fig. . Based oEvidence from TDA that IBS was linked to differences in disease severity, microbiota, and immune profiles led us to stratify the patient cohort into ME/CFS + IBS and ME/CFS without IBS and test for group-specific differences in the microbiota using linear statistical models.U test with Benjamini-Hochberg correction (adjusted p < 0.2), 21 bacterial taxa differed between the ME/CFS + IBS and control groups  and unclassified Alistipes, C. bolteae, and A. caccae (which were increased in the ME/CFS + IBS).In comparisons of the ME/CFS + IBS and control groups, LEfSe identified 12 bacterial species, genera, or families enriched in the ME/CFS + IBS and 26 bacterial taxa enriched in the controls Fig. . Based oU test with Benjamini-Hochberg correction (adjusted p < 0.2), 22 bacterial taxa differed between the ME/CFS without IBS and control groups  and unclassified Bacteroides, R. gnavus, D. longicatena, P. capillosus, E. lenta, C. symbiosum and scindens, C. bacterium, and Clostridium cf. .In the ME/CFS without IBS, LEfSe identified 29 bacterial species, genera or families enriched in ME/CFS without IBS and 16 bacterial taxa enriched in controls Fig. . Based op < 0.05, ME/CFS without IBS vs. control p < 0.001); however, only ME/CFS without IBS showed separation along PC2 . While controls had significantly lower within-group dissimilarity compared to within-group dissimilarities of both the ME/CFS without IBS and ME/CFS + IBS, ME/CFS + IBS had the highest within-group dissimilarity  Fig. . The proWe compared the number of bacterial species among the ME/CFS diagnostic groups and the controls. A total of 363 bacterial species were identified in the entire cohort. The number of species identified was similar in ME/CFS patients and controls . We also did not find differences in the number of bacterial species when comparing subgroups based on IBS status .Firmicutes phylum were the chief determinants of ME/CFS group status. The relative abundance of four bacterial species  distinguished the ME/CFS patients from the controls . The addition of four more bacterial species  improved predictive performance . This predictive selection model corresponds to our TDA, LEfSe, and nonparametric statistical findings of differences in Coprococcus, Dorea, Faecalibacterium, and Clostridium spp. in total ME/CFS and control group comparisons.We used three dimensionality reduction methods to further analyze the microbial communities that distinguish ME/CFS subgroups and controls: least absolute shrinkage and selection operation (LASSO), random forest (RF) and partial least squares (PLS) Table . BacteriFaecalibacterium cf. and Bacteroides vulgatus) distinguished the ME/CFS + IBS from the control subjects with a moderate degree of accuracy . Accuracy of the outcome prediction improved when nine additional bacterial species: F. cf., F. prausnitzii, B. vulgatus, A. putredinis, C. catus, A. caccae, D. formicigenerans, A. colihominis, and C. asparagiforme were added to the model .The relative abundance of distinct bacterial taxa defined ME/CFS patients with IBS. The relative abundance of just two bacterial species . TDA-selected bacterial species of Bacteroides, Clostridium, Pseudoflavonifractor, and Parabacteroides were found in all statistical tests  in the ME/CFS without IBS group compared to the controls. The decreased relative abundance of Bacteroides vulgatus distinguished the ME/CFS without IBS group from the ME/CFS + IBS group based on all statistical tests .Membership in the ME/CFS without IBS subgroup as compared with the control group was predicted by the relative abundance of ME/CFS patients with IBS were distinguished from ME/CFS patients without IBS by the relative abundance of eleven bacterial species .U test with Benjamini-Hochberg correction (adjusted p < 0.2) further supported findings from LEfSe showing enrichment in the pathway of atrazine degradation in both the ME/CFS and ME/CFS + IBS groups compared to the controls; predicted bacterial pathways of arginine, polyamine biosynthesis, and pyrimidine ribonucleoside degradation were reduced in the ME/CFS + IBS , ME/CFS showed altered representation of individual bacterial metabolic pathways linked to the tricarboxylic acid [Bacterial metagenomic data were used to predict differences in functional metabolic pathways in the ME/CFS subgroups. Altogether, 455 individual bacterial metabolic pathways were identified and analyzed. In superpathway analyses , LEfSe revealed that bacterial vitamin B6 biosynthesis and salvage, pyrimidine ribonucleoside degradation, and atrazine degradation were significantly enriched while bacterial pathways for the biosynthesis of arginine, polyamine, unsaturated fatty acid (FA), and mycolate were significantly reduced in the ME/CFS compared to the controls Fig. . The ME/lic acid  cycle, aImmune profiling , there was no distinct clustering observed between disease groups .We investigated whether the relative abundance of individual bacterial species, defined by their association with ME/CFS and ME/CFS subgroups  and an increased abundance of Lactonifactor and Alistipes [Firmicutes and differences from controls in representation of 40 bacterial species including F. prausnitzii, Ruminococcus spp, Coprococcus spp, E. lenta, and C. aerofaciens [Faecalibacterium species and increased Alistipes in ME/CFS as the strongest predictors for the disease.We cannot directly compare metagenomic results obtained here with others based on 16S rRNA analyses. Nonetheless, our findings replicate those of other groups in demonstrating intestinal dysbiosis in ME/CFS , 13, 22.ofaciens . Our finCampylobacter jejuni, Salmonella enterica, Shigella sonnei, Escherichia coli 0157:H7), viral (norovirus), or protozoal (Giardia lamblia) pathogens increase the risk of post-infectious IBS [The prevalence of IBS co-morbidity is high in individuals with ME/CFS 35–90%) –6. The uious IBS . ME/CFS ious IBS . Acute gious IBS . Finally–90% –6. ious IBS , 29.Faecalibacterium species, R. obeum, E. hallii, and C. comes was associated with IBS co-morbidity. The Anaerostipes genus was increased in ME/CFS + IBS, but not in ME/CFS without IBS. In contrast, the relative abundance of unclassified Bacteroides, P. capillosus, E. lenta (each increased) and P. distasonis (decreased) were identified as specific markers for ME/CFS without IBS. The relative abundance of D. longicatena was increased in ME/CFS without IBS but decreased in ME/CFS with IBS. Thus, whereas some differences in bacterial taxa in the overall ME/CFS cohort are driven by the high prevalence of IBS co-morbidity, others are specific to ME/CFS.TDA analysis revealed that IBS co-morbidity was a major driver of topological networks in our ME/CFS cohort. Decreased relative abundance of Faecalibacterium and Coprococcus species is associated with IBS-like symptoms, including colonic hypersensitivity, bloating, and GI discomfort in human and animal models [The decreased abundance of l models , 31. An l models , 32, 33.Metabolic pathways predicted from bacterial metagenomic gene content revealed additional alterations in ME/CFS and ME/CFS subgroups. Similar to our findings for differences in bacterial composition, differences in predicted bacterial metabolic pathways found in the total ME/CFS group were representative of aggregate findings associated with IBS subgroups. These results suggest that, as with bacterial taxa, some bacterial metabolic pathways may be uniquely altered in ME/CFS while others may be linked to IBS co-morbidity.Enrichment in the pathway for vitamin B6 biosynthesis and salvage was the strongest predictor of ME/CFS as well as ME/CFS without IBS, suggesting that this association is independent of IBS. Reduced functional B-vitamin status has been reported in the ME/CFS patients; however, it is unclear whether such differences can be attributed to aberrant host or bacterial metabolic pathways . PyrimidEnriched pathways for the degradation of atrazine in ME/CFS were also found in our analyses and may be independent of IBS, as the predicted pathway of atrazine degradation was a biomarker of both ME/CFS without IBS and ME/CFS + IBS. Additional studies would be needed to determine whether atrazine, a chemical found in pesticides, is present in the GI tract of these individuals and is subject to degradation by these pathways.The unsaturated FA biosynthesis pathway that was predicted to be reduced in all three ME/CFS groups is linked to energy homeostasis and basic components for several catabolic processes. The ratio of ω3/ω6 FAs and eicosapentaenoic acid/arachidonic acid are reduced in ME/CFS patients . The lowSpecific bacterial species and linked metabolic pathways were correlated with ME/CFS disease score severity . Previous studies on changes in gut microbiome following exercise challenge in ME/CFS showed alteration within 72 h in ME/CFS subjects compared to baseline and to controls . Our stuPlasma cytokines did not define ME/CFS disease groups in our cohort. Previous studies demonstrated increased pro- and anti-inflammatory cytokine levels (such as TNFα) in plasma and cerebrospinal fluid of ME/CFS patients with short duration of the disease , 48. TheOur results confirm and extend previous work indicating intestinal dysbiosis in ME/CFS. We further demonstrate that patterns of dysbiosis vary with IBS co-morbidity. Future prospective studies should consider more detailed exploration of IBS subtypes, associated GI symptoms, and their relationship to ME/CFS dysbiosis. The identification of ME/CFS networks—characterized by specific profiles that integrate microbiota, metabolic pathways, and plasma immune molecules—may enable more accurate diagnosis and lead to insights that inform the development of specific therapeutic strategies.Subjects included 50 cases and 50 controls from the Chronic Fatigue Initiative (CFI) Cohort  recruiten = 50) completed standardized screening and assessment instruments including medical history and symptom rating scales, had a physical examination, and provided simultaneous fecal and blood samples. The fecal samples were collected by subjects 24 to 48 h prior to clinical visits, stored at −20 °C, and transferred to clinical site in styrofoam boxes with ice packs provided by study coordinators. Stool and plasma materials were shipped from clinical sites to the Columbia University laboratory site on dry ice and stored at −80 °C prior to processing. Controls (n = 50) of the earlier CFI cohort study [All ME/CFS subjects n =  completeAll participants provided informed written consent in accordance with protocols approved by the Institutional Review Board at Columbia University Medical Center.Clinical symptoms and baseline health status were assessed on the day of physical examination and biological sample collection from both cases and control subjects, using two surveys: the Short Form 36 Health Survey (SF-36) and the Multidimensional Fatigue Inventory (MFI). The SF-36 includes the following subject-reported evaluations about the current health status: physical and social functioning, physical and emotional limitations, vitality, pain, general health perceptions, and mental health change . Each fu2 and normal BMI was defined as BMI <25 kg/m2. No subjects with a BMI below 18 kg/m2 (low BMI) were enrolled in our study.IBS co-morbidity was based on subject-reported diagnoses of IBS on the medical history forms. The subjects were asked if they had been diagnosed with IBS by their physician. Additional questions on the severity of symptoms and bowel frequency were included. High BMI was defined as BMI >25 kg/mSMS was carried out on DNA extracts obtained from the 100 fecal samples (50 cases and 50 controls). For Illumina library preparation, genomic DNA was sheared to a 200-bp average fragment length using a Covaris E210 focused ultrasonicator. Sheared DNA was purified and used for Illumina library construction using the KAPA Hyper Prep kit . Sequencing libraries were quantified using an Agilent Bioanalyzer 2100. Sequencing was carried out on the Illumina HiSeq 4000 platform . SMS libraries from cases and controls were grouped into 10 different pools . Each pool yielded an average of 350 million 100-bp, paired-end reads . Raw SMS data were pre-processed using prinseq (v0.20.3) for end trimming and filtered to exclude low-quality and low-complexity reads. Adaptor sequences were removed using cutadapt (v 1.8.3). Human sequences were subtracted from the dataset using bowtie2 (v2.1.0) and using genomic, mitochondrial, and ribosomal sequences downloaded from NCBI. Bacterial composition (relative abundance) was obtained from raw sequencing data using Metaphlan (v1.7.8) software and processed by Qiime (v1.8). To evaluate overall microbiome differences, we used principal coordinate analysis based on the Bray-Curtis dissimilarity metric. Metabolic pathway analysis was carried out on host-subtracted sequences using Humann2 (v0.7.1) software.TM immunoassay, Affymetrix) was used to measure plasma concentrations of immune molecules  using a standard curve derived from known reference concentrations supplied by the manufacturer. A five-parameter model was used to calculate final concentrations by interpolation. Values are expressed in pg/ml. Concentrations obtained below the sensitivity limit of detection (LOD) of the method were recoded to the mid-point between zero and the LOD for that analyte for statistical comparisons. Values obtained from reading of samples that exceeded the upper limit of the sensitivity method were further diluted and cytokine concentrations calculated accordingly. Feature scaling  was used to standardize the range of cytokine values for the MeV heatmap [A magnetic bead-based 61-plex immunoassay  using the AYASDI platform . AYASDI represents high-dimensional, complex biological data sets as a structured 3-dimensional network . Each noStandard statistical methods were applied to define the primary variables of these networks. Data were compared by nonparametric Kolmogorov-Smirnov (KS) tests to identify significant differences between networks.U test. Benjamini-Hochberg FDR  method was used to control the type I error rate at the 0.2 level [Between-group differences  in microbial composition, fecal metabolic pathway expression, plasma immune molecules, and symptom severity were tested using the nonparametric Mann-Whitney .2 level . CorrelaBacterial metagenomic and immune profiling assay data were used to develop a logistic regression model for prediction of the following binary response variables: the diagnostic groups ME/CFS, ME/CFS + IBS, ME/CFS without IBS, and controls. To eliminate potential multicollinearity, we used least absolute shrinkage and selection operation (LASSO)  and randDifferences in the relative abundance of bacteria at all taxonomic levels were determined with linear discriminant analysis effect size , which cData were analyzed and visualized with SPSS , Matlab , Prism 7 , BioVenn , and CirAdditional file 1:Table S1. (A) TDA revealed significant bacterial and metabolic pathway profile differences in ME/CFS and ME/CFS + IBS but not in ME/CFS without IBS compared to control  and individual bacterial metabolic pathways (IMPWY)). ME/CFS: myalgic encephalomyelitis/chronic fatigue syndrome, IBS: irritable bowel syndrome, KS score: Kolmogorov-Smirnov test, p: phylum, f: family, g:genus, s: species. (B) Fecal metagenomic and immune molecule profiles of ME/CFS networks based on topological data analysis. Bacterial composition, metabolic pathways, plasma immune molecules, and clinical data of ME/CFS patients separated into subgroups based on IBS co-morbidity and BMI. . Table S2. Microbial composition differences between diagnostic groups . . Table S3. Predicted bacterial metabolic pathways analyzed by Humann2 . . Table S4. Immune molecules analyzed by 61-plex immunoassay. . Table S5. Correlations of symptom severity scores with bacterial species abundance in ME/CFS and ME/CFS subgroups (ME/CFS + IBS and ME/CFS without IBS). Red indicates negative Spearman correlation; blue indicates positive Spearman correlation.Additional file 2:Figure S1. Topological data analysis of ME/CFS group. (A) The ME/CFS cases clustered into four different groups based on IBS and BMI . (B) The mean relative abundance of individual bacterial species that discriminates between the ME/CFS clusters. The mean relative abundance is indicated by the surface area of the associated circle. The discriminative changes in bacterial composition are indicated by rectangles. (C–E) Association between measures of symptom severity based on SF-36 and MFI questionnaire items and ME/CFS subgroup-associated networks (shown in A) were evaluated with TDA. (C–D) Pain and physical disability were rated as more severe  in patients with ME/CFS + IBS who had a high BMI . (E) General fatigue rankings showed greater severity in patients with ME/CFS + IBS who had a high BMI and in ME/CFS without IBS patients with a high BMI  compared to other groups. Dots that are not connected in networks represent outliers. Figure S2. Plasma immune molecule profiles of ME/CFS and controls subjects. Heatmap showing results of unsupervised hierarchical clustering based on the Euclidean distance of plasma immune molecule concentrations . The normalized concentration of immune molecules is indicated by a color scale (below heatmap) that ranges from green  through black to red . The diagnostic group corresponding to each sample is shown in the bar below the heatmap where red = ME/CFS + IBS, blue = ME/CFS without IBS, and gray = controls. (Note that immune profiles show no clear relationship with diagnostic groups.)


Campylobacter fetus is a zoonotic pathogen that occasionally causes serious, relapsing, invasive disease, especially in immunocompromised hosts. We report a case of relapsing C. fetus diarrheal illness in a 75-year-old woman which resulted in secondary bacteremia and seeding of the left knee prosthetic joint. Patient responded favorably to debridement and retention of prosthesis in addition to six weeks of meropenem followed by chronic oral doxycycline suppressive therapy. Campylobacter prosthetic joint infection (PJI) is uncommon despite the increased occurrence of arthroplasty surgeries and food-borne campylobacteriosis. C. fetus, the pathogen responsible for the majority of published cases of Campylobacter PJI, has a propensity to cause invasive infection in elderly, immunocompromised hosts, particularly those with exposure to farm animals [ C. fetus gastrointestinal disease in high-risk patients, often leading to subsequent hematogenous dissemination and PJI [ C. fetus PJI. animals . The nee and PJI . As illuA 75-year-old woman, who underwent elective left total knee arthroplasty one week prior to symptom onset, presented with acute onset of nausea, vomiting, diarrhea and worsening left knee pain of one day duration. Her medical history was significant for atrial fibrillation, pacemaker implantation for sinus node dysfunction, coronary artery disease, and insulin dependent type II diabetes mellitus. She lived on a farm by herself, denied illicit substance abuse, and had no recent travel. Three weeks prior to the left total knee arthroplasty, she was hospitalized with similar symptoms of nausea, vomiting, and diarrhea. At that time, patient was afebrile and clinically stable. Laboratory data had showed normal white blood cell (WBC) count, positive fecal leucocytes in stool, and negative cultures from blood and stool. She received symptomatic treatment with antiemetics and intravenous hydration and was discharged home the next day.∧9/L with normal differential, hemoglobin of 9.4 g/dL, platelet count of 209 × 10∧9/L, serum creatinine of 1.23 mg/dL, lactate of 1.4 mmol/L, and an elevated C-reactive protein of 244.8 mg/dL. Computerized tomography of the abdomen and pelvis did not show any acute abnormalities. X-ray of the left knee did not show effusion or fracture.Patient was asymptomatic at the time of the left knee arthroplasty surgery which was done three weeks after the first hospitalization. She had an uneventful perioperative course and was discharged to a rehabilitation facility on postoperative day 3. However, patient was hospitalized again on postoperative day 7 due to recurrence of vomiting and diarrhea associated with worsening left knee pain. Patient denied fever, chills, and abdominal pain. Stools were loose, green in color, and without blood or mucous. On physical examination, she was afebrile with stable vital signs but was lethargic and weak. Abdomen was soft and nontender with normal bowel sounds. The left knee was tender and had a well healing surgical incision without surrounding erythema, wound dehiscence, or discharge. The rest of the physical examination was unremarkable. She had a WBC count of 6.3 × 10 Campylobacter fetus subspecies fetus by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) . Patient was initially started on piperacillin-tazobactam on hospital day two, but this was later changed to meropenem. Transthoracic echocardiogram did not show any vegetation and transesophageal echocardiogram was declined by the patient. On hospital day 4, left knee synovial fluid cultures grew C. fetus. Patient underwent left knee irrigation and debridement with synovectomy and polyethylene exchange on hospital day 7. Three sets of intraoperative left knee tissue cultures grew C. fetus subspecies. fetus in sheep blood and chocolate agar plates after 48 hours. In vitro susceptibility testing was carried out using agar dilution method. The minimum inhibitory concentration (MIC) for ciprofloxacin was 1 μg/ml, meropenem, 1 μg/ml, gentamicin, 1 μg/ml, doxycycline, 0.5 μg/ml, and erythromycin, 2 μg/ml.Stool culture was repeated and resulted negative. Two sets of blood cultures from hospitalization days 1 and 3 signaled positive in the Bactec FX system  within 48 to 72 hours in the aerobic bottles only . Gram stain showed faint staining, curved Gram negative bacilli. No targets were detected on the FilmArray Blood Culture Identification (BCID) panel . After 24 hours of incubation of the positive blood culture broth in a microaerophilic environment, small, grey colonies were noted on the sheep blood agar and chocolate agar plates. The organism was identified asCampylobacter fetus” and “prosthetic joint infection” dated December 15, 2016, yielded 15 case reports published in the past three decades , there was no evidence of recurrence of infection. Campylobacter are Gram negative, curved, motile bacteria that are best known to cause food-borne diarrheal illness. C. fetus is one among 24 currently recognized species within this genus [ C. fetus is rare but may potentially be fatal, especially in the immunocompromised host [Members of the genusis genus . Human ised host , 12. C. jejuni and C. coli, C. fetus is primarily associated with extraintestinal, systemic illness which may relapse or persist for years [ C. fetus subspecies fetus is responsible for most human infections, whereas subspecies veneralis  and testudinum (which is associated with exposure to reptiles or Asian dishes made with reptiles) have rarely been associated with human disease [While the majority of gastrointestinal campylobacteriosis are caused byor years , 12. C. C. fetus is primarily a zoonotic pathogen; it colonizes the intestinal and/or genital tract of cattle and sheep, sometimes causing septic abortion in these animals [ C. jejuni/C. coli, poultry ingestion is not associated with infection since the higher body temperature of poultry birds is hostile to C. fetus [ C. fetus meningitis in a neonatal intensive care unit suggests possible human-to-human transmission [ animals . Mode of animals . Cross cC. fetus . OccupatC. fetus . A nosocsmission . In addismission . C. fetus infection depends on the host immune status and the clinical syndrome. Healthy hosts may present with culture-negative diarrheal illness [ C. fetus typically enters the blood stream from the gastrointestinal tract in susceptible hosts [The clinical manifestations of illness . Systemi illness . Our patle hosts . Bacterele hosts .C. fetus can cause endovascular infections and secondary seeding may occur in almost any body site. Infections of nervous system, lungs, abdomen, and musculoskeletal system have been reported [ C. fetus has a surface-layer (S-layer) which forms a capsule-like structure and helps the organism to evade host defense. This may explain its ability to cause a prolonged, persistent, relapsing infection for many years [reported . Infectireported . C. fetuny years . C. fetus is rarely isolated from stool culture despite the frequent occurrence of diarrhea preceding or accompanying bacteremia. This may be explained by the fact that most laboratories use selective media (which contain antibiotics) and a higher incubation temperature of 42°C to optimize growth of C. jejuni and C. coli (thermophilic species) while suppressing the growth of other fecal microbiota. C. fetus is susceptible to cephalosporins (used in some selective media) and grows variably at 42°C, thereby making its isolation from conventional stool culture difficult, and potentially causing underreporting of C. fetus diarrheal illness [ C. fetus) at an incubation temperature of 42°C.As seen in our patient, illness . For ins C. fetus infection is suspected (based on exposure history), the laboratory should be notified to perform alternative isolation techniques such as the use of less selective media, incubation at lower temperature (37°C) in a microaerophilic environment, and filtration techniques used to increase the yield of isolating C. fetus from stool culture [ C. jejuni, C. coli, and C. upsaliensis but not C. fetus. C. fetus has been isolated from commonly used aerobic blood culture bottles [If culture . The Bio bottles . If a Gr bottles . C. fetus invasive infections requires parenteral therapy; however there are no guidelines for optimal antimicrobial therapy. Resistance to ciprofloxacin, doxycycline, and erythromycin has been previously reported [ C. fetus bacteremia [ Campylobacter fetus subspecies fetus strains isolated from 1983 to 2000 in Quebec, Canada, all were susceptible to ampicillin, gentamicin, meropenem, and imipenem, with 90% minimal inhibitory concentrations of 4, 1, 0.12, and ≤0.06 μg/ml, respectively [Treatment ofreported . Fluoroqcteremia . Among 1ectively . Systemiectively . Our patC. fetus is an underrecognized and underreported pathogen which is capable of causing severe, invasive, sometimes fatal infection in humans. It may present as a chronic, persistent, relapsing infection. Alternative culture isolation techniques (different from those used for C. jejuni isolation) are needed for optimal isolation of the organism from stool. Patients presenting with febrile illness and diarrhea and who have risk factors for C. fetus bacteremia (such as residence on a farm and immunosuppression) should be evaluated for invasive C. fetus infection prior to elective arthroplasty surgery.


HOTAIR has been demonstrated to be involved in carcinogenesis and progression of various human cancers. Previous studies have investigated the potential association between HOTAIR polymorphisms and cancer risk in Chinese population. However, the results remain conflicting. Therefore, for the first time, we conducted a meta-analysis to derive a more precise estimation of these associations for Chinese. PubMed, Embase, CNKI and Wanfang databases were systematically searched. Odds ratios with 95% confidence intervals were applied to assess the association between rs920778, rs4759314, rs7958904, rs874945 and rs1899663 polymorphisms of HOTAIR and cancer susceptibility. Heterogeneity, sensitivity analysis and publication bias were conducted to measure the robustness of our findings. A total of 21 eligible studies comprising 12,278 cases and 14,532 controls were analyzed. The pooled data showed that rs920778 polymorphism was significantly associated with an increased cancer risk in all five genetic models in Chinese population. As for rs4759314 and rs874945 polymorphisms, similarly increased risks were found in specific genetic models and stratified groups. However, significant decreases in cancer risk were observed for rs7958904 in the total population, as well as in subgroup analyses. In addition, lack of association was detected between rs1899663 polymorphism and cancer susceptibility. In summary, our meta-analysis implicates possible relationship between HOTAIR polymorphisms and cancer risk in Chinese population.As a well-known long non-coding RNA,  HOTAIR, an lncRNA located on chromosome 12q13.13, is coded from the homebox C gene (HOXC) locus [HOTAIR could specifically interact with polycomb repressive complex 2 (PRC2) and LSD1/CoREST/REST complex, in turn induce its relating methylation of histone H3K27 and demethylation of histone H3K4 respectively, and consequently result in the alteration of genes expression profile [HOTAIR has been reported in a variety of human cancers such as breast cancer, gastric cancer, colorectal cancer and liver cancer [HOTAIR was also shown to be involved in the progression of multiple types of cancers, indicating that HOTAIR might serve as a useful biomarker for tumorigenesis and progression [Long non-coding RNAs (lncRNAs), as a type of transcribed RNAs which are longer than 200 nucleotides with no protein-coding capacity, were initially claimed to be a fake transcriptional noise , 2. NowaC) locus . It has  profile , 9. The r cancer –13. In agression , 14–16.HOTAIR locus have been identified [HOTAIR polymorphisms and cancer risk in Chinese population [HOTAIR with the risk of cancers including gastrointestinal cancers [HOTAIR polymorphisms and cancer risk in Chinese population. To our knowledge, this is the first meta-analysis which investigates the association for Chinese.Several single nucleotide polymorphisms (SNPs) located in entified , 18. Amopulation . From th cancers , estroge cancers –27, thyr cancers  and oste cancers . HoweverThe screening process of the studies was shown in Figure HOTAIR rs920778 polymorphism and cancer risk are presented in Table P = 0.001; TT vs. TC+CC: P = 0.018; TC+TT vs. CC: P = 0.013; TT vs. CC: P = 0.215; TC vs. CC: P = 0.041).Thirteen eligible studies including 6,854 cases and 8,477 controls were recruited in the meta-analysis. The results for the association between HOTAIR rs4759314 polymorphism and cancer risk was observed in dominant and heterozygous models for Chinese  available genotype distribution data for calculating the odds ratios (ORs) with 95% confidence intervals (CIs). Reviews, letters, conference abstracts, duplicate studies and studies without sufficient genotype information were excluded. In addition, we finally selected the study with larger sample size from duplicate publications.Studies were all selected according to the following criteria: (1) case-control design study; (2) evaluating the association between P value for Hardy-Weinberg equilibrium (HWE) in the control group. Disputes were resolved through group discussion.The following data were extracted by two independent investigators: first author, publication year, source of controls, sample size, genotype frequency, genotyping methods, age and sex matched status, type of cancers, HOTAIR polymorphisms and cancer susceptibility in Chinese population. For the rs4759314 polymorphism, the pooled ORs were estimated by allelic (G vs. A), recessive (GG vs. GA+AA), dominant (GA+GG vs. AA), homozygous (GG vs. AA) and heterozygous (GA vs. AA) models. As for rs920778, rs7958904, rs874945, and rs1899663 polymorphisms, similar five genetic models were assessed. Subgroup analyses based on source of controls, type of cancers, genotyping methods, HWE status of controls and case-control matched status were subsequently performed. HWE was examined by chi-square test in the controls. The I2 statistic was used to evaluate the heterogeneity between studies. If I2 > 50%, significant heterogeneity was found and the random-effects model should be applied. Otherwise, the fixed-effects model should be used. Sensitivity analysis was conducted by sequentially omitting each single study to evaluate the stability of our results. Publication bias was assessed by both visual inspection of funnel plot and Egger's test. The STATA software 12.0  was used for all the statistical analyses. P < 0.05 was considered to be statistically significant.The crude ORs with 95% CIs were calculated to determine the relationship between the


Escherichia coli induced infections. Using herbal medication is an alternative choice in treatment of diseases. The aim of this study was to determine the antibacterial activity of two traditionally used herbs in Iranian medicine, Anacyclus pyrethrum and Pistacia lentiscus L., on Escherichia coli.Antibiotic therapy is the main choice in treatment of Anacyclus pyrethrum and Pistacia lentiscus L. were examined in disk diffusion and skipped wells methods by measuring the diameter of inhibition zones around wells containing different concentrations of extracts from (10–1000 mg/ml) using standard broth macrodilution, method the MIC and MBC were defined.The antibacterial effect of methanolic extract of Anacyclus pyrethrum from 300 to 1000 mg/ml and the methanolic extract of Pistacia lentiscus L. from 30 to 1000 mg/ml showed antibacterial activity on Escherichia coli. The MIC of Anacyclus pyrethrum and Pistacia lentiscus L. methanolic based extract were 800 and 1000 mg/ml, respectively. The MBC was achieved at 800 mg/ml for methanolic extract of Anacyclus pyrethrum and Pistacia lentiscus L.The methanolic extract of Anacyclus pyrethrum and Pistacia lentiscus L. have antibacterial effect on Escherichia coli bacteria. This activity is dose-dependent.The methanolic extract of  Escherichia coli is a facultative Gram negative bacterium that is generally found in the lower intestine  and Pistacia lentiscus L.  are traditionally used herbs in Iranian medicine by physicians such as Avicenna (Anacyclus pyrethrum (pellitory) from Anacyclus genus is a native plant of India and Arabic countries. It has different therapeutic effects such as antmicrobial, analgesic and antioxidant activitivities  with 150μm diameter for liquor filtration. The extract was dried in Water bath at 70 C° for one week. Dried powdered extract was kept at 4°C at tightly closed vial. Different concentrations from 10 mg/ml to 1200 mg/ml in distilled water were prepared from stock solution  was obtained from the bacterial collection of Department of Microbiology, Medical Faculty, Shahed University.A swab of bacterial suspension was streaked on Muller Hinton agar plates .8 bacterial/ml and used sterile blank disks of extract dilution (McFarland 0.5 turbidity standard). After overnight 37°C aerobically condition incubation, the zones of inhibition were measured by using an Antibiotic disk Zone Reader after 24 h.The columns adjusted to 1.5 × 10The MIC was measured by broth Macro dilution test (tube Dilution) method updated by Clinical and Laboratory Standards Institute . The lowAnacyclus pyretprum had not inhibitory effect on E. coli at 10 to 200 mg/ml concentrations. From 300 to 1000 mg/ml the inhibition zone was noted.The methanolic extract of Anacyclus pyrethrum on E. coli.Anacyclus pyrethrum and Pistacia lentiscus L. have antibacterial effect on E. coli. This activity is dose-dependent.The study shows methanolic extract of E. coli such as the mixture of combining Agathosma crenulata, Dodonaea viscosa and Eucalyptus globulus from Africa and Potentilla reptans L. from Anglo-Saxon native herbs  Benth, Ageratum conyzoides Linn. From African countries and M. yunnanensis, S. sinensis, G. morella, E. daneillii, M. squamulata, S. arborescens and B. hancei from native Chinese herbal medicine have also antibacterial effect on E. coli ( E. coli –7.Anacyclus pyrethrum from 300 to 1000 mg/ml had inhibitory effect on E. coli. Based on our knowledge this is the first study on the antibacterial activity of methanolic extract of Anacyclus pyrethrum on E. coli.The study shows the methanolic extract of Anacyclus pyrethrum L. has activity against Candida albicans and Staphylococcus aureus. Oxygenated sesquiterpenes was the main effective substance (Selles et al. showed that the essential oil from Algerian ubstance .Anacyclus pyrethrum and its antibacterial effects are very rare. It has not been elucidated that which active component or either biologic mechanism of Anacyclus pyrethrum has antibacterial activity on E. coli.Studies on biologically active substance of Pistacia lentiscus L. was studied more than Anacyclus pyrethrum. Some studies on this field were focused on oral cavity bacteria (Pistacia lentiscus L. has antibacterial effecton Staphylococcus aureus, Bacillus subtilis, Prevotella melaninogenica and Klebsiella pneumoniae (The antibacterial activity of bacteria –20. It heumoniae –24.Pistacia lentiscusL. Escherichia coli, Staphylococcus aureus, and Bacillus subtilis have different sensitivity to these compounds (The alpha-Pinene, beta-myrcene, beta-pinene, limonene, and beta-caryophyllene are major antibacterial components of essential oil and gum of ompounds .Pistacia lentiscus L. have different effects on different bacteria. Mharti et al. demonstrated that germanicol, thunbergol, himachalene, trans-squalene, terpinyl propionate, 3,3-dimenthol and cadina-1.4-diene derived from essential oil of the leaves of Pistacia lentiscus L. have antibacterial effect on Klebsiella pneumoniae, but not on Pseudomonas aeruginosa (It seems that different components of ruginosa .Anacyclus pyrethrum and Pistacia lentiscus L. have antibacterial effect on E. coli. More researches on identifying the effective components of Anacyclus pyrethrum and Pistacia lentiscus L. on Escherichia coli are necessary.The present study shows that methanolic extract of Anacyclus pyrethrum and Pistacia lentiscus L. have antibacterial effect on Escherichia coli bacteria. This activity is dose-dependent.In conclusion, the methanolic extract of


Alternanthera philoxeroides, with its co-occurring non-invasive functional counterparts, the native congener Alternanthera sessilis, the exotic Myriophyllum aquaticum and the native Jussiaea repens, in three manipulative substrates with different soil distribution patterns. We found that the invasive performance of A. philoxeroides was not enhanced by heterogeneity and that it was generally scale independent. However, A. philoxeroides showed some advantages over the three non-invasives with respect to trait values and phenotypic variation. These advantages may enhance the competitive capacity of A. philoxeroides and thus promote its invasion success in heterogeneous environments.Environmental heterogeneity is considered to play a defining role in promoting invasion success, and it favours clonal plants. Although clonality has been demonstrated to be correlated with the invasion success of several species of clonal invasive plants in heterogeneous environments, little is known about how the spatial scale of heterogeneity affects their performance. In addition, the factors that distinguish invasive from non-invasive clonal species and that enhance the invasive potential of clonal exotic invaders in heterogeneous environments remain unclear. In this study, we compared several traits of a noxious clonal invasive species,  Environmental heterogeneity plays a significant role in biological invasion12346791014Trait comparison of co-occurring invasive and non-invasive species to reveal the competitive advantages of invasives over non-invasives generally focuses on two aspects related to invasion mechanisms – trait values and trait plasticity17192010131415Trait plasticity refers to the property of phenotypic variation of a genotype in variable environments1724252627232931Alternanthera philoxeroides. Three non-invasive wetland stoloniferous clonal plant species that co-occur with A. philoxeroides – a native congener Alternanthera sessilis, an exotic non-invasive Myriophyllum aquaticum and the native Jussiaea repens – were selected as comparison species to explore the invasion mechanism of A. philoxeroides in heterogeneous environments. Alternanthera philoxeroides competes with the three non-invasive counterparts as these four species occupy similar ecological niche in the field . The following hypotheses were postulated: i. A. philoxeroides benefits from soil heterogeneity, and the benefit strength is patch-scale dependent. ii. Compared with the three non-invasive functional counterparts, the invasive A. philoxeroides displays some trait advantages in the presence of different soil distribution patterns.In this study, we investigated the relationship between clonal plant invasiveness and environmental heterogeneity in a manipulative experiment. The correlation between clonal performance and the spatial distribution of soil was studied in a stoloniferous weed species that is a detrimental invasive in many regions worldwide, – Alternanthera philoxeroides is a stoloniferous clonal herb with a strong dispersal ability in terrestrial, semi-aquatic and aquatic habitats2534A. sessilis is mainly distributed in moist habitats such as swamps, wetlands and the edges of ditches and canalsAs a stoloniferous clonal herb with sexual reproduction, A. philoxeroides, M. aquaticum survives in both aquatic and semi-aquatic habitatsOriginating from South America as Jussiaea repens is a dominant native species occurring in sub-tropical and tropical regions in ChinaA. philoxeroides, A. sessilis and J. repens and 10-cm-long shoots with approximately 14 leaf whorls for M. aquaticum) were collected of each species from mono-populations of each species in the riparian zone of Liangzi Lake . All of the collected plant materials were pre-cultivated in sandy clay three times for approximately two months before the experiment set-up. Twenty-four morphologically identical plants without branches were then selected of each species. Six plants of each species were randomly selected and dried to determine the initial biomass. The eighteen remaining plants  were selected for the experiment.On June 25, 2015, fifty clonal ramets sharing similar morphology  and sand ; (2) heterogeneous substrate, composed of two contrasting patches of equal volumes of clay and sand (He1); and (3) heterogeneous substrate with six adjoining patches of equal volumes of clay and sand (He2) (−1) was maintained above the substrate surface throughout the experimental period. The experiment was established on August 30, 2015.Seventy-two round basins  were selected as mesocosms. Three substrate types were designed: (1) homogeneous substrate (Ho), comprised of a homogenous mixture of equal volumes of clay  . All of rd to 5th leaf node on the apical end of the stolon were selected to gauge leaf area  except for the leaves of M. aquaticum. As the leaves of M. aquaticum are tiny and needle-like, leaf area could not be precisely measured by the leaf area metre. Therefore, five leaves on the 5th to 7th leaf node on the apical end of the stolon were randomly selected and scanned  to produce 1:1 high definition images  on a whiteboard with a ruler. Then, ImageJ 1.46  was used to analyse the leaf area. The selected leaves of the four species were oven dried at 70 °C for 72 h to determine dry biomass. Specific leaf area (SLA) was calculated as followsAll of the plant materials were harvested on November 25, 2015, after 87 days of growth. Immediately after harvest, stolon length was measured and ramet number was counted for each replicate. Five leaves of each replicate on the 3The mean SLA values of the selected five leaves of each replicate were used for data analysis.A. sessilis) for each replicate. The biomass of each plant part in each replicate was determined after oven drying at 70 °C for 72 h. Total biomass, relative growth rate (RGR), leaf mass ratio (LMR), stolon mass ratio (SMR), root mass ratio (RMR) and root-shoot ratio (R/S ratio) were calculated as followsThe remnant parts of the plant materials were separated into leaves, stolon and roots .All of the data met the assumptions of normality and homogeneity of variance prior to analysis. Stolon length, ramet number and total biomass were each transformed using the functions of log(log(x)), log(x) and log(x + 1), respectively. RGR, SMR and RMR were transformed using the square root(x) function. R/S ratio was transformed using the xThe species exerted significant effects on all traits, and the substrate type exerted significant effects on all traits except SLA . SignifiAlternanthera philoxeroides showed similar trait values of stolon length, SLA, total biomass, RGR, RMR and R/S ratio among the different substrates  lower values of stolon length and RGR, respectively, in the heterogeneous substrates than in Ho  lower values of stolon length, ramet number, total biomass, RGR and SMR, respectively, in heterogeneous substrates than in Ho  higher values of stolon length (P < 0.001), total biomass (P = 0.010), RGR (P < 0.001), RMR (P < 0.001) and R/S ratio (P < 0.001), respectively, than A. sessilis on average (P = 0.078) and LMR (P = 0.249) were shared between A. philoxeroides and A. sessilis on average (Alternanthera philoxeroides showed 11.3% and 9.52% (significantly) lower values of SLA (P = 0.014) and SMR (P < 0.001), respectively, than A. sessilis on average  lower values of stolon length (P < 0.001), SLA (P < 0.001), total biomass (P = 0.001), RGR (P = 0.017) and LMR (P < 0.001), respectively, than M. aquaticum on average (A. philoxeroides and M. aquaticum on average (P = 0.349)  higher values of SMR (P < 0.001), RMR (P < 0.001) and R/S ratio (P < 0.001), respectively, than M. aquaticum on average ( average . Similar= 0.349) . Alterna average .Alternanthera philoxeroides showed 28.4%, 45.0%, 10.2%, 57.9%, 24.2% and 49.6% (significantly) lower values of stolon length (P < 0.001), ramet number (P < 0.001), SLA (P = 0.033), total biomass (P < 0.001), RGR (P < 0.001) and LMR (P < 0.001), respectively, than J. repens on average (A. philoxeroides and J. repens on average (P = 0.299) (Alternanthera philoxeroides showed 13.5% and 9.2% (significantly) higher values of RMR (P = 0.002) and R/S ratio (P = 0.002), respectively, than J. repens on average ( average . Similar= 0.299) . Alterna average .Glechoma hederacea and patch size. Moreover, Zhou et al.A. philoxeroides gained some benefits from soil heterogeneity regardless of patch size. First, A. philoxeroides recruited more ramets in heterogeneous substrates than in the homogeneous substrate  in A. philoxeroides than in M. aquaticum and J. repens likely enabled A. philoxeroides to integrate the heterogeneity more efficiently and thus enhance its invasive potential. In contrast, the maladaptive trait plasticity shown by M. aquaticum and J. repens may confer fitness costs from an evolutionary perspective31Overall,  average . In termogeneity . Second,bstrates . InconsiA. philoxeroides. This conclusion is based on our finding of approximately consistent performance maintenance across different soil distribution patterns. However, some advantages of A. philoxeroides over its non-invasive co-occurring functional counterparts with respect to trait values and phenotypic variation may help explain the successful invasion of the noxious clonal weed A. philoxeroides in heterogeneous environments. Additionally, based on our observations, we predict that soil texture, temporal scale and growing season are likely to influence the growth of the four evaluated species. Future studies should focus on how diversified environmental heterogeneity due to various ecological factors affects the invasive performance of clonal alien species.In summary, soil heterogeneity is unlikely to be a primary promoter of invasive success in How to cite this article: Wang, T. et al. The invasive stoloniferous clonal plant Alternanthera philoxeroides outperforms its co-occurring non-invasive functional counterparts in heterogeneous soil environments - invasion implications. Sci. Rep.6, 38036; doi: 10.1038/srep38036 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Tick-borne diseases are the most prevalent vector-borne diseases in Europe. Knowledge on the incidence and clinical presentation of other tick-borne diseases than Lyme borreliosis and tick-borne encephalitis is minimal, despite the high human exposure to these pathogens through tick bites. Using molecular detection techniques, the frequency of tick-borne infections after exposure through tick bites was estimated.Ticks, blood samples and questionnaires on health status were collected from patients that visited their general practitioner with a tick bite or erythema migrans in 2007 and 2008. The presence of several tick-borne pathogens in 314 ticks and 626 blood samples of this cohort were analyzed using PCR-based methods. Using multivariate logistic regression, associations were explored between pathogens detected in blood and self-reported symptoms at enrolment and during a three-month follow-up period.Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Candidatus Neoehrlichia mikurensis, Rickettsia helvetica, Rickettsia monacensis, Borrelia miyamotoi and several Babesia species. Among 92 Borrelia burgdorferi s. l. positive ticks, 33% carried another pathogen from a different genus. In blood of sixteen out of 626 persons with tick bites or erythema migrans, DNA was detected from Candidatus Neoehrlichia mikurensis (n = 7), Anaplasma phagocytophilum (n = 5), Babesia divergens (n = 3), Borrelia miyamotoi (n = 1) and Borrelia burgdorferi s. l. (n = 1). None of these sixteen individuals reported any overt symptoms that would indicate a corresponding illness during the three-month follow-up period. No associations were found between the presence of pathogen DNA in blood and; self-reported symptoms, with pathogen DNA in the corresponding ticks (n = 8), reported tick attachment duration, tick engorgement, or antibiotic treatment at enrolment.Half of the ticks removed from humans tested positive for Based on molecular detection techniques, the probability of infection with a tick-borne pathogen other than Lyme spirochetes after a tick bite is roughly 2.4%, in the Netherlands. Similarly, among patients with erythema migrans, the probability of a co-infection with another tick-borne pathogen is approximately 2.7%. How often these infections cause disease symptoms or to what extend co-infections affect the course of Lyme borreliosis needs further investigations. Two most common tick-borne diseases in Europe are Lyme borreliosis and tick-borne encephalitis. Ticks transmit many more pathogens, causing neglected diseases such as anaplasmosis, babesiosis, rickettsiosis and neoehrlichiosis. These diseases are seldom diagnosed, due to their mild and non-characteristic symptoms, but also due to lack of awareness and availability of diagnostic tests. Using molecular detection techniques (polymerase chain reaction or PCR), we estimated the frequency of tick-borne infections in humans after a tick bite and in patients with the first symptoms of Lyme borreliosis, an erythema migrans. About half of the ticks that fed on humans carried one or more tick-borne pathogens, and approximately 2.5% of people that were bitten by ticks were infected with a tick-borne pathogen other than Lyme borreliosis or tick-borne encephalitis. Co-infections of a tick-borne pathogen in patients with an erythema migrans was also approximately 2.5%. Based on these findings, we estimated the incidence of tick-borne infections other than Lyme borreliosis in the Netherlands. How often these infections cause disease or to what extend co-infections affect the course of Lyme borreliosis needs further investigations. Borrelia burgdorferi sensu lato complex [Lyme borreliosis is the most prevalent tick-borne disease in humans, and is caused by spirochetes of the  complex –3. The m complex –3. Tick- complex .Ixodes ricinus ticks transmit several Borrelia burgdorferi s. l. genospecies, but are also infected with a variety of established or potentially pathogenic microorganisms, such as Borrelia miyamotoi, Anaplasma phagocytophilum, Candidatus Neoehrlichia mikurensis, several Babesia species, Rickettsia helvetica, R. monacensis and TBEV [I. ricinus ticks carrying B. burgdorferi s. l. are co-infected with one or more pathogens from a different genus [In several European countries, there have been marked increases in the incidence of Lyme borreliosis and TBE over the past ten to twenty years –7. In thand TBEV –15. Thesnt genus , implyinB. miyamotoi disease in an immunocompromised patient in 2012 [B. burgdorferi s. l. with different tick-borne pathogens may possibly contribute to the high variety of clinical manifestations that are associated with Lyme borreliosis.Remarkably, little is known about the incidences and clinical presentations of other tick-borne diseases than Lyme borreliosis and TBE. In general, disease caused by these other tick-borne pathogens, are associated with febrile illnesses with fever, headache, myalgia and malaise –21. Howe in 2012 . It has  in 2012 –28. TherSeveral reasons can be appointed for the absence in reporting of tick-borne diseases other than Lyme borreliosis and TBE, and the diagnosis of co-infections with other pathogens in Lyme borreliosis patients. Firstly, most of these infections might be self-limiting without overt or characteristic symptoms, often a clear-cut case definition of patients infected with one of these pathogens has not been established yet. Secondly, a poor performance or non-existence of supportive laboratory tests in routine medical microbiological settings. Thirdly, the lack of awareness among health professionals.B. burgdorferi s. l. can be shown in patients with early localized Lyme borreliosis and in people exposed to tick bites in the Netherlands, and to determine ii) the clinical picture of patients with DNA of tick-borne pathogens in their blood.Here, we aim to investigate i) whether infection with tick-borne pathogens other than Our approach is to test for the presence of nucleic acid (DNA/RNA) of the specific pathogens in human blood through amplification with PCR, especially since currently; there is no other specific laboratory diagnostic to detect infection with most of these tick-borne pathogens. Compared to DNA amplification with PCR, available serological tests generally have a low specificity and or sensitivity, particularly during the early phase of infection. In addition, although culturing is considered the most reliable method in proving the presence of microorganisms, it is time consuming, costly and often not possible for all pathogens.I. ricinus were identified. At enrolment, two tubes of blood were collected, 7 ml in a serum tube and 5 ml in an EDTA tube. Three months after enrolment, follow-up questionnaires and a consecutive 7 ml serum sample was collected from the tick bitten patients and from the EM patients after standard antibiotic treatment [Ticks, EDTA-blood and questionnaire data were available from a nationwide prospective observational study among patients who consulted one of 307 enrolling general practitioners for a tick bite or EM between May 2007 and December 2008 in the Netherlands, as described in detail . All parreatment , 30. SevBorrelia-, Ehrlichia-, Anaplasma-, Rickettsia- and Babesia-species. Further identification by DNA sequencing was performed as described [B. garinii and B. bavariensis [B. miyamotoi in ticks was tested by a real-time PCR amplification in 302 ticks, and Candidatus Neoehrlichia mikurensis in 312 ticks. The presence of TBEV RNA could not be screened in the tick samples, since only DNA had been extracted from these samples. Individual test results of the tick analyses were not reported to the participants or their physicians, in accordance with the informed consent form.After arrival at the laboratory, ticks were stored at –20°C in ethanol. DNA was extracted using the DNeasy Blood and Tissue Kit  according to the manufacturer’s instructions extraction for ticks. After total DNA extraction from ticks and amplification by PCR, reverse line blotting (RLB) was performed for escribed , 31. PCRariensis . The preB. burgdorferi s. l., B. miyamotoi, A. phagocytophilum, Candidatus Neoehrlichia mikurensis, spotted fever Rickettsia's carried out on a LightCycler 480 . For primers and probes, see Babesia genospecies, we performed a conventional PCR targeting the 18S rRNA gene on all the blood samples [Extraction of whole nucleic acid of the EDTA-blood samples were performed using robot-extraction  from 400 μL of EDTA-plasma  according to the manufacturer’s instructions in a diagnostic laboratory setting. All 626 samples were analyzed with different real-time PCRs based on various genes specific for the microorganism of interest namely;  samples , followeThe prevalence of microorganism DNA detection in ticks and in EDTA-blood was calculated with 95% confidence intervals (95%CI) based on mid-P exact. Characteristics of persons with or without DNA detected in blood by PCR were compared in Chi-square or Fisher’s exact test. We looked for associations between DNA detected in EDTA-blood by PCR and DNA detected in available ticks from the participants, tick engorgement, patient-reported tick attachment duration, antibiotic treatment at enrolment, and patient-reported symptoms at enrolment and after three months. Using multivariate logistic regression, we explored for associations between DNA detected in blood by PCR and self-reported symptoms at enrolment and follow-up. All reported clinical symptoms (at enrolment and follow-up) were included as predictive variables in the multivariate logistic regression models, after which the models were optimized using backwards elimination, until all predictive variables that were maintained in the model were statistically significant contributors (p<0.05). Statistical analyses were performed with SAS 9.4 (SAS Inc.).Borrelia burgdorferi s. l. DNA was detected in 92 (29%) ticks, as published earlier [Candidatus Neoehrlichia mikurensis (5.4%), A. phagocytophilum (1.0%), Rickettsia species (22%), Babesia species (3.5%). and B. miyamotoi (2.3%). DNA of microorganisms of two or more genera were detected in 34 ticks (11%). Among the 92 B. burgdorferi s. l. positive ticks, 30 (33%) also carried a pathogen of a different genus. About half of the ticks  tested negative for all genera.ed in 92 % ticks, B. burgdorferi s. l. and one (0.2%) for B. miyamotoi in the real-time PCRs multiplex, both with high Ct values. In another multiplex, five blood samples (0.8%) were positive for A. phagocytophilum and seven (1.1%) for Candidatus Neoehrlichia mikurensis. Three (0.5%) blood samples for Babesia genospecies yielded a sequence in conventional PCR, in which genetic analyses showed to be B. divergens. None of the samples were found positive for spotted fever Rickettsia's or TBEV.Candidatus Neoehrlichia mikurensis sequence yielded a partial groEL sequence and five out of seven could also be confirmed on a separate gene, namely gltA. The seven groEL are 100% identical to each other as were the five gltA sequences  up to 18.8% (R. helvetica). With an estimated incidence of 1.1 million tick bites per year, human exposure to a tick-borne pathogen other than B. burgdorferi s. l. and TBEV varies from roughly 3500 for B. divergens, and 3500 for R. monacensis to 207,000 persons for R. helvetica. Among the 322,000 persons exposed to B. burgdorferi s. l. through a tick bite, roughly 105,000 are simultaneously exposed to another pathogen. In addition, exposure to more than one tick-borne pathogen can occur when people have more than one tick bite at once or several consecutive tick bites.The tick samples were screened by a different method (RLB) than the blood samples . In 314 ticks removed from humans a wide variety of tick-borne pathogens were detected namely, s before –43. The B. burgdorferi s. l. after tick bites was estimated to be 5.1% [Candidatus Neoehrlichia mikurensis, A. phagocytophilum, B. divergens, B. miyamotoi and B. burgdorferi s. l. DNA in the blood of sixteen individuals after exposure to a tick bite. None of these cases reported to be immunocompromised, and all the EM patients were treated with antibiotics according to the guidelines for treatment of Lyme borreliosis [Clearly, not all exposure to tick-borne pathogens results in human infection. Based on the development of an EM or seroconversion, the risk of infection with  be 5.1% . In thisreliosis . Mild clCandidatus Neoehrlichia mikurensis infection was observed in 1.1% (95%CI 0.5%–2.2%). Candidatus Neoehrlichia mikurensis infections have been described in immunocompromised patients [Anaplasma phagocytophilum infection was found in 0.9% (95%CI 0.3%–2.0%) of the persons exposed to tick bites in the Netherlands  of the persons exposed to tick bites. In Europe, only two cases of human babesiosis have been reported in immune-competent patients, one due to B. divergens [B. miyamotoi 0.2% (95%CI 0.0%–0.8%). The presence of B. miyamotoi DNA could only be determined by real-time PCR, and several attempts to confirm this finding by conventional PCR was unsuccessful. This patient had received antibiotic treatment at enrolment for his EM, so a low bacterial load in blood due to the treatment could be an explanation for the high Ct value. Evidence for infection with B. miyamotoi in Europe comes from one immunocompromised case [In this study, patients , and morpatients –48. Anapherlands . Geneticcotype I . Evidencse cases , 49. Thesymptoms . Babesiaivergens . Only onsed case , and a ssed case .A. phagocytophilum and B. divergens. The severity of self-reported symptoms of the seven EM patients with a co-infection was indistinguishable from patients only having EM. No indications were found that infection with a tick-borne pathogen other than B. burgdorferi s. l. caused overt symptoms that would indicate a corresponding illness. The low number of persons with a tick bite or EM that were identified with an tick-borne pathogen infection other than B. burgdorferi s. l., in combination with the limited medical assessments, and the used method of pathogen detection are not sufficient to infer how often tick-borne pathogens other than B. burgdorferi s. l. (and TBEV) cause disease. Also, to what extend they affect the diagnoses and the etiology of Lyme borreliosis. Furthermore, the ability for a pathogen to cause disease depends also on extrinsic factors for example the immune status of its host.Altogether, the probability of infection with a tick-borne pathogen other than Lyme spirochetes after tick bites in the Netherlands is roughly 2.4% (95%CI 1.1%–4.5%). This number is similar to the probability of a co-infection with another tick-borne pathogen in patients with EM . Interestingly, one patient in this study had a co-infection with B. burgdorferi s. l. and TBEV, and their ability to cause infection in the general population, warrants increased awareness, knowledge, improvement of diagnostic tests and a clear-cut clinical case definitions in an European setting. Only when better laboratory tests are available for these tick-borne diseases, their impact as a co-infection with Lyme borreliosis can be assessed.The high exposure to tick-borne pathogens other than S1 Checklist(DOC)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Mycobacterium bovis) than for M. tuberculosis TB. We compared treatment response times for patients with M. bovis TB and M. tuberculosis TB reported in the United States during 2006–2013. We included culture-positive, pulmonary TB patients with genotyping results who received standard 4-drug treatment at the time of diagnosis. Time to sputum-culture conversion was defined as time between treatment start date and date of first consistently culture-negative sputum. We analyzed 297 case-patients with M. bovis TB and 30,848 case-patients with M. tuberculosis TB. After 2 months of treatment, 71% of M. bovis and 65% of M. tuberculosis TB patients showed conversion of sputum cultures to negative. Likelihood of culture conversion was higher for M. bovis than for M. tuberculosis, even after controlling for treatment administration type, sex, and a composite indicator of bacillary burden.Current US guidelines recommend longer treatment for tuberculosis (TB) caused by pyrazinamide-resistant organisms (e.g.,  Mycobacterium tuberculosis complex is composed of several genetically related and pathogenic mycobacterial species, including M. tuberculosis and M. bovis  genotyping techniques  at the Centers for Disease Control and Prevention  and restricted analysis to cases reported during 2006–2013 to permit sufficient time for follow-up reporting of outcome data. M. tuberculosis or M. bovis were excluded; cases with isolates identified as M. bovis Bacillus Calmette–Guérin were assumed to be iatrogenic  at diagnosis  or subsequent positive cultures. We used a Kaplan-Meier estimator to calculate survival-like function curves for time to sputum-culture conversion for persons with We censored, at the date recorded for each outcome, patients who died during treatment and those who moved, were lost to follow-up, or otherwise stopped treatment before the expected completion date; we restricted the analysis to the first 90 days of anti-TB treatment. We used Cox proportional hazard modeling to calculate adjusted hazard ratios (aHRs) with 95% CIs of factors associated with time to culture conversion. We tested the proportional hazards assumption by graphing log  versus log (time) for all covariates of interest. We assessed a priori covariates of interest, including sex; treatment administration type (directly observed therapy versus self-administered therapy); reported HIV status ; sputum smear status ; and cavitary disease found by diagnostic imaging  results.After reviewing smear status and diagnostic imaging results, we created a composite variable for bacillary burden  to avoid collinearity. We categorized cases with confirmed cavitary disease identified by imaging and positive sputum smear as high bacillary burden, cases with either confirmed cavitary disease or positive sputum smear as medium bacillary burden, cases with no cavitary disease and negative sputum smears as low bacillary burden, and cases without any of these indicators as unknown bacillary burden. In the time-to-event analysis, we excluded patients who had unknown bacillary burden. Analysis was conducted by using SAS version 9.3 .M. tuberculosis TB or M. bovis TB. All cases of TB with initial resistance to isoniazid or rifampin  and M. tuberculosis TB cases with any initial resistance to pyrazinamide (n = 757) were excluded. The final dataset for analysis included 297 cases of M. bovis TB and 30,848 cases of M. tuberculosis TB. All covariates met the proportional hazards assumption except for HIV status, which was excluded from further analysis because of sparse data.A total of 91,985 TB cases during 2006–2013 were available for analysis . ApproxiM. bovis TB and patients given a diagnosis of M. tuberculosis TB were similar  than patients with M. tuberculosis TB . Patients with M. bovis TB were most often born outside the United States (86.2%), and most self-identified as Hispanic (93.2%). In comparison, just over half (59.5%) of M. tuberculosis TB patients were born outside the United States; 30.3% self-identified as Hispanic (p<0.0001 for both comparisons).The age distributions of patients given a diagnosis of  similar . A greatM. bovis TB patients had pulmonary and extrapulmonary involvement at diagnosis compared with 10.0% of M. tuberculosis TB patients (p<0.0001). Similar proportions of patients had cavitary chest lesions documented  and positive sputum smear results . Among M. tuberculosis TB patients, proportions categorized as high bacillary burden (36.2%) and medium bacillary burden (37.1%) differed significantly from those for M. bovis TB patients  (p = 0.03).Approximately one third (35.0%) of M. bovis TB patients and 65% of M. tuberculosis TB patients showed conversion of their sputum cultures to negative (p<0.01)  relative to M. tuberculosis TB patients, after controlling for treatment administration type, sex, and the composite indicator for bacillary burden  . By the negative . On the y burden . DirectlM. bovis TB than for patients with M. tuberculosis TB after controlling for treatment administration type, sex, and a composite indicator of bacillary burden. This finding was not documented previously and is especially intriguing because M. bovis is inherently resistant to pyrazinamide, a first-line anti-TB drug used during the first 2 months of treatment and credited with reducing relapse rates of the 6-month regimen to levels similar to the 9-month regimen without this drug  than M. tuberculosis TB patients. We also found similar proportions of cavitary lesions on chest imaging, which might indicate a similar pathogenicity between organisms when pulmonary disease is present. However, this finding could not be directly studied because NTSS does not collect data on smear grade. Alternatively, differential gene expression, proinflammatory macrophage response, growth in macrophages, and lipid profiles might have roles in explaining variability in bacillary populations between species  and drug susceptibility testing are not readily available  or drug susceptibility testing results . Thus, some patients with M. bovis genotyping results or pyrazinamide resistance might have received a different regimen at some point after the start of treatment. NTSS does not capture information on changes to regimens during the course of treatment or the date when drug susceptibility testing results were received, and we were unable to assess this information directly.We attempted to control for potential variations in treatment by including only patients initially given a standard 4-drug regimen . However, some clinicians might have changed regimens after receiving genotyping results . Thus, for 50% of cases, clinicians would have received genotyping results after the event time of this analysis , which would diminish any potential influence on our findings. Moreover, removing pyrazinamide from regimens used to treat Concurrent and immunosuppressive conditions, such as diabetes mellitus, end-stage renal disease, and hematologic or reticuloendothelial malignancies, which might influence time to culture conversion, were not routinely collected during our study period. These variables were included as part of routine reporting in 2009 and might be helpful in future studies  as in our adjusted model  died during treatment. This finding suggests that earlier culture conversion does not necessarily lead to better clinical outcomes. Further laboratory studies should be conducted to better monitor and assess the time to sputum-culture conversion and clinical outcomes between these 2 M. tuberculosis complex species. If similar results are observed, further randomized clinical trials for treatment duration (and possibly treatment regimen) might be warranted.Our findings should be interpreted with caution. Although we found earlier culture conversion for


Streptomyces sp. TP-A0648 isolated from a leaf of Aucuba japonica. This strain produces a new tumor cell growth inhibitor designated anicemycin. The genome harbors at least 12 biosynthetic gene clusters for polyketides and nonribosomal peptides, suggesting the potential to produce diverse secondary metabolites.We report the draft genome sequence of  Streptomyces sp. TP-A0648 from a leaf of Aucubajaponica Thunb. collected in Toyama prefecture, Japan and discovered anicemycin as an inhibitor of anchorage-independent growth of human ovary cancer SKOV-3 cells from its culture broth. Anicemycin consists of four partial structures adenine, aminosugar, glycine, and fatty acid . The whole genome of Streptomyces sp. TP-A0648 was read using a combined strategy of shotgun sequencing with GS FLX+  and pair-end sequencing with MiSeq . These reads were assembled using Newbler v2.8, and subsequently finished using GenoFinisher (S. griseus NBRC 13350 (Type I PKS gene clusters in scaffold00001 and scaffold00020 showed high sequence similarities to biosynthetic gene clusters for annimycins  and C-10RC 13350 . The remStreptomyces sp. TP-A0648 to produce diverse polyketides and nonribosomal peptides. Further detailed inspection of the genome data may disclose the biosynthetic genes for anicemycin, for which the biosynthetic mechanisms remain unknown.The genome analysis suggests the potential of Streptomyces sp. TP-A0648 has been deposited in the DDBJ/ENA/GenBank database under the accession no. BDHJ00000000. The version described in this paper is the first version, BDHJ01000000.The draft genome sequence of


S. porci, S. porcorum and S. plurextorum associated with pneumonia and septicemia. Although these three species showed similar MALDI profiles, several peaks were recognized that can be useful for their differentiation: S. porci , S. plurextorum  and S. porcorum . After adding these MSPs, an evaluation was conducted to determine the accuracy of MALDI-TOF MS for the identification of streptococci from diseased pigs using 74 field isolates. Isolates were identified as S. suis, S. porcinus, S. dysgalactiae, S. hyovaginalis, S. porcorum, S. alactolyticus, S. hyointestinalis and S. orisratti. This is the first time that the latter three species have been reported from clinical specimens of pigs. Overall, there was good concordance (95.9%) between the results obtained from MALDI-TOF MS identification (best hint) and those from genotyping. Our results demonstrate the good performance of MALDI-TOF MS (100% sensitivity and specificity) for identifying most of the species of streptococci that can frequently be isolated from diseased pigs. However, conflicting results were observed in the correct identification of some isolates of S. dysgalactiae and S. alactolyticus.The application of MALDI-TOF MS for identifying streptococcal isolates recovered from clinical specimens of diseased pigs was evaluated. For this proposal, the MALDI BDAL Database  was supplemented with the main spectrum profiles (MSP) of the reference strains of  Streptococcus suis, Streptococcus dysgalactiae subsp. equisimilis and Streptococcus porcinus are well-recognized swine pathogens [S. suis is by far the most important agent of infectious diseases in this group. It is associated with a variety of clinical conditions including meningitis, septicemia, arthritis, endocarditis and pneumonia. It has also been isolated from cases of rhinitis and abortion. S. dysgalactiae subsp. equisimilis causes sporadic cases of septicemia and arthritis in sucking pigs, endocarditis in growing pigs and ascending infection of the uterus in sows [S. porcinus is the etiological agent of streptococcal lymphadenitis in growing pigs. It also causes throat abscesses and has sometimes been isolated from pneumonia [Streptococcus species have been isolated from clinical specimens in swine. Streptococcus plurextorum has been isolated from lesions associated with pneumonia and septicemia [Streptococcus porci and Streptococcus porcorum have also been associated with pneumonia in pigs [Several species of streptococci, such as athogens , 2. S. s in sows , 3. S. pneumonia , 4, 5. Opticemia  and Stre in pigs , 8. HoweStreptococcus species is traditionally based on phenotypic/biochemical techniques. However, identifying the Streptococcus species using these tests can be difficult [S. suis [S. plurextorum, S. porci and S. porcorum are species that are phylogenetically closely related to S. suis but they have not been evaluated yet by MALDI-TOF MS. Therefore, the aim of the present work was to incorporate the main spectrum profiles (MSP) of reference strains of S. porci, S. porcorum and S. plurextorum into the MALDI BDAL Database  and then explore the usefulness of the MALDI-TOF MS technology as a diagnostic tool for the identification of streptococci associated with infections in pigs.Routine identification and differentiation of the ifficult , 10. Theifficult . This teifficult –14. In p[S. suis . Howeverhttp://asforce.org/course/assets/img/module2/transport.pdf). Upon arrival, clinical samples were processed in biological safety cabinets within 12h. Samples were cultured on Columbia and Columbia-CNA agar plates that were incubated at 37°C for 24h under aerobic conditions.The present work does not include any experimental infection trials with pigs. Only diseased farmed pigs were used to microbiologically identify the etiological agent of the disease. We did not, therefore, consult with the Institutional Animal Care and Use Committee and no specific national regulations for these procedures are available. Diseased pigs were necropsied under strict hygienic conditions by the veterinarian in the farms immediately after the pig´s death and clinical specimens were sent to the Health Surveillance VISAVET Centre of the Universidad Complutense  for a confirmatory microbiological diagnosis. Clinical specimens were handled following the recommendations of the OIE for the transport of specimens of animal origin (S. plurextorum (1956-02T and 1355–03), S. porci (2923-03T and 2857–03), and S. porcorum (682-03T and 1792–03). These strains were identified during polyphasic taxonomic studies that guaranteed their correct identification. These taxonomic studies included biochemical, genetic and phylogenetic analysis of the isolates [A first set of strains was used to create the Main Spectrum Profiles (MSP) to be included in the Bruker MALDI BDAL database : 5′-agagtttgatcctggctcag-3′) and pH* (5′-aaggaggtgatccagccgca-3′) and the amplified product was further sequenced bidirectionally [http://eztaxon-e.ezbiocloud.net/). All the strains were grown on Columbia sheep agar plates at 37°C under aerobic conditions for 24h and single colonies were selected for MALDI-TOF MS analysis. For S. suis isolates, identification was determined by a S. suis species-specific PCR [gdh).After the MSPs of these species were defined and incorporated into the MALDI- BDAL Database , a second set of 74 additional field isolates was used to validate the utility of the MALDI-TOF MS approach for the diagnosis of diseases in pigs caused by these bacteria. Isolates were originated from clinical samples of diseased pigs . MALDI-Ttionally . The 16Sific PCR  that ampCells from representative bacterial colonies were re-suspended in 300μL of HPLC-grade water and mixed vigorously prior to 900μL of HPLC-grade ethanol being added. Subsequently, an acetonitrile/formic acid extraction protocol was performed in accordance with the manufacturer’s instructions . After protein extraction, one microliter of each isolate extract was spotted onto a 384-spot polished steel target plate, let to dry at room temperature and overlaid with one microliter of α-cyano-4-hydroxy-cinnamic acid (HCCA) matrix. Data acquisition was conducted in linear positive ion mode using a Bruker Daltonik UltrafleXtreme MALDI TOF/TOF system with a set mass range of 2-20KDa and a nitrogen laser (335 nm) for ionization of the molecules. The specific settings for spectra acquisition were: laser frequency: 2000 Hz, acceleration voltage: 25 kV. Each spectrum was acquired using FlexControl software (Version 3.4) in automatic mode in a random sampling pattern.S. plurextorum, n = 2; S. porci, n = 2 and S. porcorum, n = 2) were entered manually in our MALDI database, which included all the Bruker MALDI BDAL database entries plus three entries of S. suis serotypes 2, 7 and 9 as described previously [S. plurextorum, S. porci and S. porcorum, a total of 24 spectra per isolate were obtained from the 8 spots in three measurements. Spectra were analyzed by FlexAnalysis  according to [S. porcorum, S. porci and S. plurextorum were evaluated using FlexAnalysis software.Six new entries . The reliability of the identification was evaluated according to the log (score) values, calculated with the MALDI Biotyper software mentioned above. Interpretation of the score value obtained for each isolate was performed according to the manufacturer’s instructions: values between 3.000–2.300 indicated highly probable species identification, values from 2.299 to 2.000 indicated secure genus and probable species identification, while lower scores between 1.999 and 1.700 were considered as probable genus identification. The Biotyper software showed a total of 10 top matches (defined by the user) in an identification ranking list ordered according to the score value of each identification.After inclusion of the new MSPs, a panel of 74 The identification accuracy of MALDI-TOF MS  for each streptococcal species under study was estimated using WINPEPI (PEPI-for-Windows) v. 11.35 .S. porci and S. plurextorum were quite similar, but closer analysis of their mass peak profiles revealed some different mass patterns for each species  the isolates of these species gave identification score values lower than 1.900, which indicates these species would be clearly differentiated from any of the Streptococcus species currently included in the mentioned database, including those species associated with disease in pigs. After the MSPs of these streptococcal species were incorporated into the Bruker MALDI BDAL database, the utility of MALDI-TOF MS for identifying those species of streptococci associated with disease in pigs was evaluated with 74 streptococcal field isolates recovered from different clinical specimens of diseased pigs . Specificity values for S. alactolyticus and S. dysgalactiae were also 100%, but the sensitivity values were 0% (95% CI: 0–35.43) and 37.5% (95% CI: 13.68–69.43), respectively, considering erroneous identifications  and those with inconsistent identifications .The results of MALDI-TOF MS identification (best hint) were concordant in most isolates with that of genetic identification . More than half of the isolates  showed score values higher than 2.300, while the remaining isolates  presented scores between 2.299–2.000, values considered as probable identification at species level . Evaluatications  those isS. suis, S. porcinus, S. hyointestinalis, S. hyovaginalis, S. porcorum and S. orisratti as all isolates of these species were identified with score values >2.0 while was the second identification option gave always score values of <2.0  at the time of this study. However, the species S. porci, S. porcorum and S. plurextorum associated with pneumonia and septicemia in pigs [S. porci, S. porcorum and S. plurextorum and insert them into the Bruker database. Analysis of the MSPs of these three species as well as those of S. suis, S. dysgalactiae and S. porcinus identified some species-specific m/z peaks that exhibited good discriminatory power based on the data of their intensity, signal-to-noise and frequency of detection  were identified as S. suis, S. porcinus or S. dysgalactiae, a result that is in line with the epidemiological relevance of these pathogens in the swine industry [S. hyointestinalis, S. alactolyticus and Streptococcus orisratti was also interesting Click here for additional data file.