External auditory exostoses (EAE) have been noted among the Neandertals and a few other Pleistocene humans, but until recently they have been discussed primary as minor pathological lesions with possible auditory consequences. An assessment of available western Eurasian late Middle and Late Pleistocene human temporal bones with sufficiently preserved auditory canals (n = 77) provides modest levels of EAE among late Middle Pleistocene archaic humans (≈20%) and early modern humans . The Neandertals, however, exhibit an exceptionally high level of EAE . The levels of EAE for the early modern humans are well within recent human ranges of variation, frequencies which are low for equatorial inland and high latitude samples but occasionally higher elsewhere. The Early/Mid Upper Paleolithic frequency is nonetheless high for a high latitude sample under interpleniglacial conditions. Given the strong etiological and environmental associations of EAE development with exposure to cold water and/or damp wind chill, the high frequency of EAE among the Neandertals implies frequent aquatic resource exploitation, more frequent than the archeological and stable isotopic evidence for Middle Paleolithic/Neandertal littoral and freshwater resource foraging implies. As such, the Neandertal data parallel a similar pattern evident in eastern Eurasian archaic humans. Yet, factors in addition to cold water/wind exposure may well have contributed to their high EAE frequencies. In his classic monograph on the Neandertal skeleton from the Bouffia Bonneval in La Chapelle-aux-Saints, Corrèze, France, Marcellin Boule  noted thExternal auditory exostoses  are dense bony growths protruding into the external auditory canal. They are frequently observed clinically in modern humans, often in the context of aquatic sports (hence references to their presence as “swimmer’s ear” or “surfer’s ear”). First documented more than a century ago clinically ,12, theiHowever, EAE may occur in a variety of contexts, given sufficient inflammation of the soft tissue lining of the auditory canal. They are often benign but can lead to cerumen impaction and conductive hearing loss ,26,36–39Given this framework, observations on EAE among the available western Eurasian Middle and Upper Paleolithic humans, including both presence/absence and degree of formation, are provided. The primary samples of concern are Middle Paleolithic Neandertals and Upper Paleolithic modern humans, but observations are also included for the few sufficiently intact late Middle Pleistocene archaic humans and early Late Pleistocene  modern humans.External auditory exostoses are identified as growths into the auditory canal from its squamous and/or tympanic portions ,22,46. TThe EAE were scored using the four part ordinal scale of Cooper et al. , Crowe eA number of the Pleistocene tympanic bones are thickened at their lateral margins, especially where the inferior tympanic crest meets the porus, and some of them exhibit small nubbins of bone along the lateral tympanic margin. These changes are not considered to be part of the EAE, which consist of bony growths into the meatus.The paleontological observations  have beeThe principal methodological limit of the study is the difficulty in scoring early EAE see ,32), and, and32])The primary samples include Neandertals and earlier and later Upper Paleolithic modern humans . The Nea2 p = 0.976). They are also biased towards young adults plus older adolescents versus older adults (2 p = 0.941). Therefore, although EAE, as a degenerative process, generally increases in frequency and/or severity with age and/or exposure [The three primary samples are all male biased (for the sexable specimens) , althougr adults , as are exposure ,43,49–51To provide a recent human reference framework, presence/absence data are presented for 120 recent human samples . As origIn addition, to provide a framework for EAE severity distributions, the fossil scores are compared to the available grade data for a smaller number (14) of recent human samples ,42,51,52In the statistical comparisons, exact Chi-square and binomial p-values were computed using StatXact 4.0.1 , and WilAll of the later Pleistocene western Eurasian samples provide at least one individual with clearly developed external auditory exostoses. In the two smaller samples, the late Middle Pleistocene archaic human and the Middle Paleolithic modern human ones, one individual in each sample provides a modest (grade 1) EAE, providing sample frequencies of 20% and 25%  of 5.1%– 71.6% for the late Middle Pleistocene sample and 6.3%– 80.6% for the Middle Paleolithic modern human one). If the values have any meaning given the large CIs, they suggest frequencies among the higher of the recent human middle latitude and low latitude “wet” samples .The larger Upper Paleolithic samples provide frequencies with smaller 95% CIs, ones of 20.8% (CI: 4.8%– 48.5%) for the E/MUP sample and 9.5% (CI: 1.2%– 30.4%) for the LUP sample . Their 9In contrast, the Neandertal sample, with a mean frequency of 56.5% (CI: 34.5%– 76.8%) is well above all except two low latitude “wet” samples, one each from the Canary Islands and the southern Brazilian coast . The nexThe presence/absence frequencies for EAE are reflected as well in the distributions of the severity grades across the samples . The EAEEven though there is little difference across the primary samples in terms of younger versus older individuals, it is possible that some of the more developed Neandertal EAE is partially age-related. The only Late Pleistocene individual with grade 3 exostoses, Shanidar 1, is an older adult based on dental wear and pubic symphyseal morphology . The oldIt is possible to evaluate the degree of sexual difference in EAE presence for the Neandertal and E/MUP samples, despite the small sample sizes per sex; the other samples are too small or have too few EAE cases to provide a reliable assessment. In the Neandertal sample, for the 12 individuals for which sex can be assigned or estimated, EAE occurs in 60.0% of the males (n = 8) and 75% of the females (n = 4). In the 19 sexable E/MUP specimens, EAE are present in 16.7% of males (n = 12) and 28.6% of females (n = 7). The females have modestly higher frequencies in both samples, but in neither case are the sex frequencies significantly different from 0.5 . The pattern of higher male frequencies in many recent human samples therefore does not appear to be present among these Late Pleistocene humans.In her 1986 analysis of global distributions of EAE, Kennedy  argued tTherefore, as discussed above (see esp. ), there In this context, the frequency of EAE in the LUP sample is unexceptional relative to recent humans. It is low relative to the middle latitude “wet” samples, and moderately high for the low latitude “dry” and the high latitude samples. Among the sites, Moča, Oberkassel and Zlatý kůn are modestly north of 45° N , and the southern French, northern Italian and Swiss sites distribute around the 45° N latitude. Only the Sicilian site of San Teodoro and the Israeli one of Ohalo II are below 40°. It nonetheless remains unclear how much colder these sites may have been during MIS 2 . The onlThe E/MUP sample provides a higher overall frequency of EAE, albeit all grade 1. The sites yielding observable auditory meatus range from 44° to 56° N, and the five specimens with EAE on at least one side span this geographic range (from Cioclovina 1 at 45° to Sunghir 1 at 56°). Of these specimens, only one (Bausu da Ture 2) can be considered coastal , even atIf the E/MUP EAE frequency is moderately high for the relevant recent human environmental group, the Neandertal one is exceptional. As noted, only six of the recent human samples have frequencies above its 95% CI lower limit, and only two “wet” samples provide frequencies similar to the Neandertal one. The Neandertal sample spans middle to high latitudes, from 33° to 50°. Only three of the specimens can be considered as coastal  –70, althEvidence for aquatic resource exploitation among these Neandertals varies considerably, in part due to older excavations  at a number of the sites yielding human remains with sufficiently intact temporal bones. There is skeletal and residue analysis evidence for the exploitation of fish among Neandertals in western Europe –76, caseAlthough there is no evidence of littoral resource exploitation in the region of Tabun Cave, there is evidence of mollusk use further north on the eastern Mediterranean coast ,86. TherIn addition, although most of the EAE present in Neandertal auditory canals are characteristic rounded, knob-like growths , three oThere is therefore a substantial decrease in EAE frequency from Neandertals to early modern humans in western Eurasia, with a possible decrease from the earlier to later Upper Paleolithic modern humans. The Upper Paleolithic samples are nonetheless not significantly different from each other despite a lower frequency in the LUP sample (see 95% CIs above); the Neandertal and LUP frequencies are significantly different, and the Neandertal and E/MUP frequencies are modestly less distinct (if not significantly different).2 p = 0.238). The LUP sample is intermediate in its latitudinal distribution , it has the highest number of specimens with maritime/riverine/lacustrine contexts (9 of 21), but it also has the lowest EAE frequency. It is therefore possible that factors other than cold water exposure influenced the differences between the Neandertals and the Upper Paleolithic modern human samples. This inference is supported by the apparent increase in aquatic resource exploitation through the Late Pleistocene, of both littoral and riverine/lacustrine resources [The E/MUP sample is significantly higher in latitude than especially the Neandertal one (Wilcoxon p < 0.001), but they do not differ meaningfully in terms of maritime/riverine/lacustrine versus inland contexts ; the addition of the three early Late Pleistocene archaic humans raises the frequency to 68.8% (n = 18) [These data from western and eastern Eurasia therefore suggest that etiological factors in addition to cold water irritation of the auditory canal may have contributed to their elevated frequency of this condition. Could general meatal sanitary conditions be responsible for the additional growths? Could many of these individuals have had a predisposition to develop exostoses in the auditory canal, at higher frequencies than among most recent human populations? Or are the external auditory exostoses indicating a pattern of aquatic resource foraging prior to the Upper Paleolithic that is poorly documented in the Eurasian archeological and stable isotopic records?Building on the previously scattered observations of external auditory exostoses among the Neandertals, it appears that there was a very high frequency of EAE among these late archaic humans, yet unexceptional levels of them (relative to most recent human samples) among western Eurasian early modern humans. The frequency of EAE for the E/MUP sample is nonetheless moderately high for a sample that should have been paleoclimatically similar to recent human high latitude (>45°) populations. It remains likely that the high level of EAE among the Neandertals, and the E/MUP samples given its paleoenvironmental context, is due in part to the exploitation of aquatic resources. However, the Neandertal frequency is at the upper limits of recent human population values and is matched only by those who experienced cold water maritime climates. It is therefore likely that, as with eastern Eurasian later archaic humans, multiple factors were involved in their abundance of EAE.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.


The greatest impact on profitability of a commercial beef operation is reproduction. However, in beef heifers, little is known about the vaginal and fecal microbiota with respect to their relationship with fertility. To this end, we followed heifers through gestation to examine the dynamics of vaginal and fecal microbial composition throughout pregnancy.n = 72), during the first (n = 72), and second trimester (n = 72) for all individuals, and third trimester for individuals with confirmed pregnancies (n = 56). Fecal samples were taken at pre-breeding (n = 32) and during the first trimester (n = 32), including bred and open individuals. Next generation sequencing of the V4 region of the16S rRNA gene via the Illumina MiSeq platform was applied to all samples. Shannon indices and the number of observed bacterial features were the same in fecal samples. However, significant differences in vaginal microbiome diversity between gestation stages were observed. No differences in beta-diversity were detected in vaginal or fecal samples regarding pregnancy status, but such differences were seen with fecal microbiome over time. Random Forest was developed to identify predictors of pregnancy status in vaginal  and fecal  samples.Heifers were exposed to an estrus synchronization protocol, observed over a 12-day period, artificially inseminated 12 h to 18 h after observed estrus, and subsequently exposed to bulls for a 50-day breeding season. Vaginal samples were taken at pre-breeding (Our study shows that bovine vaginal and fecal microbiome could be used as biomarkers of bovine reproduction. Further experiments are needed to validate these biomarkers and to examine their roles in a female’s ability to establish pregnancy. Reproduction has the greatest impact on profitability in commercial beef operations . The losLactobacillus species [Lactobacillus dominance can lead to the overgrowth of pathogenic bacteria resulting in bacterial vaginosis (BV) which is associated with adverse pregnancy outcomes [Aggregatibacter, Streptobacillus, Cronobacter, Phocoenobacter and Psychrilyobacter [Lactobacillus species in the vaginal ecosystem is low at less than 3.5% and 0.53% for chimpanzees and ewes, respectively [The extensively characterized human vaginal microbiome is divided into 5 community state types (CSTs) based on the dominating species of bacteria –8. Four  species , 10. In outcomes , 11–13. outcomes . The eweyobacter . Unlike ectively , 15.Parvimonas, Porphyromonas, unclassified Veillonellaceae, Mycoplasma and Bacteroidetes [Aggregatibacter, Streptobacillus, Phocoenobacter, Sediminicola and Sporobacter species are reported in a study by Swartz et al. [Ruminococcus, Dialister, Aeribacillus, and Porphyromonas were dominant colonizers in a study reported by Gonzalez and colleagues [Histophilus has also been isolated from bovine vaginal communities diagnosed with a reproductive disorder, and not from those possessing a healthy reproductive function [In cattle, various studies report a variety of microbial compositions related to the vagina in female bovine. Laguardia-Nascimento et al.  characteroidetes . Dominanz et al. . Memberslleagues . Differelleagues . Histophfunction .Lactobacillus dominance is crucial to vaginal health in humans, but not other species [The interrelationship between hosts and their microbes is important in female fertility. In humans and non-human species alike, the suppression and over colonization of certain bacterial species in a niche results in disease pathogenicity and emphasizes the importance of understanding the interaction between host environment and its inhabiting microbes microbes –23. It h species . Studies species . When in species . UnderstMany steps will be needed to draw a causal relationship between vaginal microbiome and reproductive traits. Some of these steps include a first discovery type study to show the correlation, the isolation and re-inoculation of certain bacteria of interests into the vagina or the reduction of these bacteria to see the changes in reproductive traits. Therefore, the purpose of this study is the first step towards this end, to characterize the vaginal microbiome of commercial beef heifers and to determine if the vaginal microbiome could be used to predict the ability of a heifer to establish a pregnancy. Furthermore, this study seeks to understand the dynamic communities of vaginal environments in the gestating heifer by following individuals throughout pregnancy. Due to the important role that gut microbiome plays , we alsoAll animal work was approved and all methods were performed in accordance with guidelines of the Institutional Animal Care and Use Committee of the University of Arkansas under protocol # 16024. The University of Arkansas Division of Agriculture’s Beef Research Unit near Fayetteville, AR, housed 72 crossbred beef heifers averaging 420.88 ± 17.42 d of age and 328.036 ± 25.45 kg at the initiation of this study.At the onset of the breeding season, a 25 mg PGF2α injection  was administered intramuscularly in the neck. A heat detection patch  was placed on the rump of each female. Heifers were then allocated to 1 of 6, 1 ha grass pastures. Each day for the subsequent 7 d, all heifers were monitored for estrus activity at 8:30 a.m. and 4:30 p.m.. Within 12 h to 18 h of detected estrus, heifers were artificially inseminated .Following day 7 of estrus detection, those individuals not showing signs of estrus like behavior were administered a second PGF2α injection. This group of heifers were monitored and artificially inseminated as described above for 5 additional days. The remaining heifers were then moved to 6, 2.4 ha fescue-bermuda grass mixed pastures and were rotated every 28 d. Seven days after transfer to the pastures, a fertile bull was introduced to each pasture to initiate a 50-day breeding season. The bulls were rotated among the pasture every 7 d. A breeding soundness examination was performed on each bull no greater than 30 d before introduction to the heifer herd and following the 50 d breeding season to confirm fertility. After 50 d of exposure all bulls passed breeding soundness examinations.Ultrasound was performed twice: on 63 d to determine pregnancies resulting from artificial insemination and on 60 d after bull removal to determine pregnancies resulting from bulls, when it is easy to identify AI versus bull bred pregnancies due to difference in crown-rump length  was retracted into the outer guard and the double guarded swab was removed from the animal. The swab was cut from the handle, placed in a 2-mL snap-cap tube with 1 mL of AMIES transport buffer and placed on ice. All samples were stored at −80 °C. Fecal and vaginal samples were taken from all individuals, as described previously at a second time point during the first trimester of gestation. Vaginal swabs were also taken from all heifers during the second trimester of gestation and again for those with confirmed pregnancies during the third gestational trimester  at a 45Detailed health records were maintained for each heifer throughout the entirety of the trial to ensure the health status of each individual. Each female was vaccinated with an inactivated vaccine containing IBR (BHV), BVD, BRSV, PI3, Leptospirosis and Vibriosis and treated for external and internal parasites according to the University of Arkansas Division of Agriculture’s Beef Research Unit cattle management protocol. Upon completion of the trial, pregnant heifers were maintained as one group and open heifers were culled. The retained females grazed on fescue-bermuda grass pastures and were supplemented with adequate free choice mineral supplements during gestation. Within 24 h of birth, calf sex and birthweight were recorded .Approximately 0.1 g of thawed feces was used for DNA extraction using the QIAamp PowerFecal DNA Kit  according to the manufacturer’s protocol. DNA was extracted from the vaginal swabs using the QIAAmp BiOStic Bacteremia DNA Kit  according to the manufacturer’s protocol. Nanodrop One C  was used to measure the DNA concentration and purity.n = 272; fecal, n = 64) were combined. The exact size of the library product and the concentration was measured with a KAPA Library Quantification Kit  through quantitative PCR  assay and an Agilent 2100 Bioanalyzer System . The library was diluted based on the results from the qPCR and the bioanalyzer [For library preparation, 10 ng of DNA were used for PCR amplification targeting the V4 region of the 16S rRNA gene. PCR was used to amplify each sample using dual index primers according to . Amplicov/v) (Illumina) was added to the mix and 600 μL were loaded into a MiSeq® v2 (500 cycle) reagent cartridge for sequencing. The sequencing procedure was monitored periodically throughout the assay using the Illumina BaseSpace® website.The 20 nmol/L pooled library, containing 336 individual samples, and a PhiX control v3 (20 nmol/L) (Illumina) were mixed with 0.2 N NaOH and HT1 buffer (Illumina). PhiX control v3  (approximately 250 base pairs in length) were downloaded to a local computer from the Illumina BaseSpace® website and the data was processed using the Deblur program integrated in the QIIME2 pipeline , 30. Deb  and fecal (n = 64) were utilized for DNA extraction and sequencing of the V4 region of the 16S rRNA gene. After removing low quality reads and chimeras using QIIME 2 (2018.8), 3,617,919 and 1,584,626 high quality reads remained for vaginal and fecal samples respectively. Vaginal samples averaged 13,862 reads per sample ranging from 1153 to 98,623 . Microbial diversity (Shannon index) from both animals with and without established pregnancies increased from pre-breeding to the second trimester . Consistently, no significant differences in the total number of observed OTUs were observed in fecal samples . P values for pairwise comparison of fecal samples are presented in Table At the community level, although remarkable variation in the inter-animal dynamics of the alpha diversity was observed  between the open and the bred female cattle were observed at any time point.No significant differences in fecal or vaginal alpha diversity measures , but differences in community membership changed based on time . The Bray-Curtis index was used to estimate dissimilarities in both community membership and structure. PCoA plot based on Bray-Curtis distance shows no distinct clustering according to pregnancy status or time. No differences based on pregnancy status were seen , but differences in community structure were observed based on time .We next examined dissimilarities in community membership and structure between pregnant and non-pregnant females overtime. The Jaccard dissimilarity matrix was used to evaluate bacterial community membership. To visualize the Jaccard distances, principal coordinate analysis (PCoA) was applied to the Jaccard dissimilarity matrix. Vaginal samples representative of all time points and each pregnancy status cluster together on principle coordinate axes 1 and 2 . No differences based on pregnancy status were detected . No differences based on pregnancy status were observed . Consistently, significant differences in fecal community structure over time was demonstrated in the PCoA plot based on Bray-Curtis distances . Similar patterns were observed based on the PCoA plots based on unweighted UniFrac  and an unclassified Bacteroidaceae  using the AUC-RF algorithm. For the vaginal microbiome, 15 features from the pre-breeding vaginal samples selected by random forest were able to predict if a cow could become pregnant with an AUC of 0.849  and the Shannon index being 1.4 to 4.1 times greater than those without BV [Lactobacillus species play in the bovine vaginal ecosystem is yet to be determined, but it is possible that other species dominating the bovine vaginal niche have similar function.No significant differences in bacterial community richness were observed in vaginal or fecal samples comparing bred and open females. In humans , 41, 4 onaerobes , 8. Bactnaerobes . In a huLactobacillus related OTUs are enriched in women with increased gestational age explaining changes in community membership and structure in late gestating humans [Human vaginal microbiome studies have demonstrated the development of a more stable vaginal microbiota near the end of the gestation period. Aagaard et al.  reportedg humans .Interestingly, although significant differences in bovine fecal microbial membership and structure over time were detected, no change in community membership or structure was observed in the vaginal niche of the female bovine throughout gestation, suggesting the bovine vaginal microbiome was stable and not affected by any of these factors. In addition, no changes or clusters were observed to differentiate pregnant from non-pregnant females in either vaginal or fecal samples at the community level.Escherichia/Shigella, and Ureaplasma. Escherichia has been documented as a contributing pathogen to metritis (uterine inflammation) due to its ability to establish residency in the reproductive tract from contamination by feces, ascend up the reproductive axis and maintain a presence in a contaminated uterus [Escherichia and reproductive failure [Ureaplasma is a common isolate from cervicovaginal mucosal samples from beef females with healthy reproductive tracts [Ureaplasma has been associated with cows suffering from granular vulvitis syndrome and mastitis, ciliostasis in cultured oviductal tissues and humans experiencing reproductive failure and infertility [U. diversum in combination with Pasteurella and/or Manheimia species causes lung lesions in calves resulting in pneumonia and consequent reoccurring morbidity [Ureaplasma isolation in vaginal samples, and since it’s presence is similar among bred and open females, this could explain a requirement for interaction with other pathogens to cause disease.In this study, the top 2 dominating features of the vaginal microbiota are affiliated with d uterus , 44. In  failure . Ureaplae tracts . Howeverertility . A previorbidity . This stHistophilus somni was listed as the #1 predictor of pregnancy. Rodrigues and colleagues described the vaginal microbiome of female cattle with reproductive disorder. They found Bacteroides, Enterobacteriaceae, and Histophilus to be the top 3 dominant OTUs in unhealthy animals. Based on Random Forest predictors from this study, Histophilus can be used to predict the pregnancy status in vaginal samples before breeding. Histophilus species are gram-negative, non-spore-forming bacterium that can exist in both pathogenic and non-pathogenic forms [H. somni are isolated from the bovine mucous membranes of nasal passages, the prepuce and sheath of males and in the vagina of females [Histophilus in females that do not establish a pregnancy in vaginal samples agrees with the presence of this OTU in animals with reproductive disorder and suggests the potential role that Histophilus plays in causing reproductive disorder. Clostridiaceae was observed in vaginal and uterine samples of animals and humans [Campylobacter, a clinical human and animal pathogen, can cause bovine venereal campylobacteriosis or vibriosis, which is the primary cause of abortion and infertility in cattle [Campylobaacter were also found as one of the most important vaginal bacteria causing abortion in sheep [Campylobacter, was listed as #3 predictor by Random Forest to predict pregnancy. This bacterium was only present in female cattle that did not establish pregnancy, which agrees with the reported infertility in heifers caused by Campylobacter infection.It is of particular interests to predict the likelihood of a heifer to establish a pregnancy based on her vaginal microbiome for improved reproduction strategies. Using Random Forest, we were able to identify 15 bacterial features that accurately AUC = 0.849) differentiate heifers which established pregnancy from those that never did at the pre-breeding stage. Member of  differend humans , 50, 51.d humans , 51, butn cattle , 53. In in sheep . Feature2 and H2 [Interestingly, bovine fecal microbiome predicted the establishment of pregnancy with even a higher accuracy than the vaginal microbiome (AUC = 0.929) with just 15 bacterial features. These features are associated with Bacteroidales, Ruminococcaceae, Coriobacteriaceae. Coriobacteriaceae has been isolated from the vagina of cattle with and without reproductive disorder, but it’s function is more accurately described in its symbiotic relationship with the gut of insects , 55. Thi2 and H2 , fecal [2 and H2 , 58, mil2 and H2  vaginal 2 and H2  of dairy2 and H2  showed t2 and H2 . A recen2 and H2 , which mIn conclusion, the ability to use the vaginal microbiome in beef heifers to predict reproductive potential and gestational period is confirmed. Using Random Forest we identified specific bacterial strains that can predict pregnancy status, for both vaginal and fecal niches. Findings from this study advance the knowledge of the microbial communities residing in the vagina of beef heifers before breeding and throughout pregnancy.Additional file 1: Figure S1. Flow chart of the experimental design and sample collection.Additional file 2: Figure S2. Rarefaction curve. (A) Rarefaction plot of vaginal samples (one curve per group). (B) Rarefaction plot of fecal samples. 1, 2, 3 and 4 in figure legend represent the pregnancy stage of pre-breeding, first trimester, second trimester, and third trimester, respectively.Additional file 3: Figure S3. Alpha diversity (Shannon index) change of vaginal microbiota for each sample. The bottom and top of each box are the first and third quartiles, respectively, and the band inside the box is the median. The connected point represent alpha diversity of animal vaginal samples at different stage.Additional file 4: Figure S4. Principal Coordinates Analysis (PCoA) of unweighted UniFrac distances in vaginal (A) and fecal (B) samples across gestation stages and between open and bred cattle. Triangles and circle represent bred and open females, respectively. Stages are indicated by color: red, blue, green and purple represent pre breeding, and gestational trimesters 1 through 3 respectively. These stages were defined based on the status of the pregnant cattle. Open cattle stayed open throughout the whole experiment. Samples were collected prospectively but pregnancy was defined retrospectively. The ellipses were calculated and drawn with 0.95 of confidence level. Bred: cattle that were pregnant after the breeding season; Open: cattle that never established pregnancy.Additional file 5: Figure S5. Relative abundance of vaginal microbiota at phylum level for each sample with different pregnancy status. Multi-colored stack bar graphs represent the relative abundance. Each chart represents a stage .Additional file 6: Figure S6. Relative abundance of fecal microbiota at phylum level for each sample with different pregnancy status. Multi-colored stack bar graphs represent the relative abundance. Each chart represents a stage .Additional file 7: Figure S7. Relative abundance of predictive bacterial features in vaginal samples across gestation stages and between open and bred cattle. 1, 2, 3 and 4 On the X-axis represent the pregnancy stage of pre-breeding, first trimester, second trimester, and third trimester, respectively.Additional file 8: Figure S8. Relative abundance of predictive bacterial features in fecal samples at pre-breeding and first trimester and between open and bred cattle. 1, 2, 3 and 4 On the X-axis represent the pregnancy stage of pre-breeding, first trimester, second trimester, and third trimester, respectively.Additional file 9: Table S1. Sequencing results of mock community.Additional file 10: Table S2. Summary of sequencing and alpha diversity results for fecal and vaginal samples.


Two artisanal varieties of cheese made in Spain, one made of ewes’ raw milk and the other of goats’ raw milk were selected to evaluate the effect of a high hydrostatic pressure (HHP) treatment at 400 MPa during 10 min at 2 °C on the formation of biogenic amines (BA). These conditions were applied at the beginning of the ripening  and in the case of ewes’ milk cheeses also after 15th days (HHP15). BA formation was greatly influenced by HHP treatments in both types of cheese. HHP1 treatments significantly reduced the amounts of BA after ripening, being tyramine and putrescine the most affected BA in goats’ milk cheeses and tyramine and cadaverine in ewes’ milk cheeses. The BA reduction in the HHP1 samples could be explained by the significant decrease in microbiological counts, especially in the LAB, enteroccocci and enterobacteria groups at the beginning of ripening. The proteolysis in these samples was also affected reducing the amount of free amino acids. Although proteolysis in ewes’ milk cheeses HHP15 was similar than in control samples a reduction of BA was observed probably because the decrease caused on microbial counts. Biogenic amines (BA) are basic nitrogenous compounds formed in different foodstuffs due to the microbial decarboxylation of amino acids. The kind and the amount of BA formed depends on the decarboxylase capability of the bacterial strains present, the availability of substrate amino acids and the physicochemical properties of the matrix . Some arDuring cheese manufacture several factors may contribute to the accumulation of toxic amounts of BA. Good manufacturing practices to minimize the occurrence of BA-producing microorganisms in raw materials, pasteurization of milk or addition of BA-non-producing starter cultures have been suggested as BA risk mitigation options. Although it has been described that milk pasteurization reduces the level of decarboxylase-positive bacteria, later contamination of milk and curd during cheese manufacturing by decarboxylating bacteria and their subsequent growth and metabolic activity during cheese ripening usually results in BA build up ,11,12,13High hydrostatic pressure (HHP) is non-thermal processing method used to extend shelf-life of foods. This induces morphological changes and inhibition of enzymes and genetic mechanisms of microorganism . HHP offThe main objective of this survey was to evaluate HHP processing to reduce BA formation in two varieties of artisanal Spanish cheese made of raw milk from ewe and goat. Treatments were applied at different stages of ripening and the consequences of these treatments on the BA formation and proteolytic activity were evaluated during the ripening.Two types of artisanal ripened cheeses elaborated in Spain were studied in this work, both made with starter culture, enzymatic curd and pressed paste. The first one was produced from goats’ raw milk in the region of Catalonia, northeast of Spain, and the second was made from ewes’ raw milk in Castilla y León, central Spain. Three independent batches of each type of cheese were produced following the usual manufacturing procedures used by the manufacturers.HHP treatments were performed at 400 MPa for 10 min at a temperature of 2 °C using an Alstom HHP equipment  with a 2 L pressure chamber. A mixture of alcohol and water (1:9) was used into the chamber. The pressurization rate and depressurization time were 268 MPa/min and 55 s, respectively. Before processing, goats’ milk cheese samples were separated in two batches: samples not HHP treated (Control samples) and samples HHP treated before the 5th day of ripening (HHP1). In the case of ewes’ milk cheese samples a third batch for samples that were treated after 15 days of ripening (HHP15) was included. In all cases a portion of about 8.0 cm diameter was obtained and shaped to adequate it to the diameter of the cylinder of the HHP equipment and then vacuum packaged. After the HHP treatments, samples were deprived of the plastic bag and kept into a ripening chamber at 14 °C and 88% of relative humidity to continue with the ripening process for 60 days. Analyses of cheeses were performed at the 5th, 15th, 30th, 45th and 60th day of ripening.Lactococcus spp. were made on M-17 agar (Oxoid) supplemented with a bacteriological grade lactose solution  and incubated at 30 °C for 48 h; Lactobacilli were determined on Man Rogose Sharpe agar  and incubated at 30 °C for 48 h; Enterococci were enumerated using KF Streptococcus Agar (Oxoid) supplemented with 2,3,5-trifeniltetrazolium chloride solution 1% (Oxoid) and incubated at 37 °C for 48 h; enumeration of Enterobactericeae was performed on Violet Red Bile Glucose Agar  and counts of Escherichia coli was made on the chromogenic selective media Coli ID  and incubated at 30 °C for 24 h; Staphylococcus aureus counts were determined on Bair Parker Agar supplemented with rabbit plasma fibrinogen  and incubated at 37 °C for 24–48 h.Ten grams of each sample were homogenized in 90 mL of sterile Buffered Peptone Water  with a paddle blender . Counts of WSN) fraction was obtained and determined by the Dumas combustion method [WSN fraction was expressed as a percentage of Total Nitrogen (TN), described as the Ripening Index (RI), according to the formula:Water Soluble Extracts (WSE) of cheese were prepared according to the method described by Kuchroo and Fox . From thn method . The nitl-Leucine per g of cheese.The measurement of the amino group content was determined on WSE by the Trinitrobenzensulphonic Acid method (TNBS) according to the procedure described by Hernández-Herrero et al. . ResultsThe RP-HPLC method described by Eerola et al.  and modip < 0.05. Comparisons of mean values of proteolysis indexes were performed using the Student-Newman-Keuls test with the significance level set at p < 0.05. All tests were performed with the SPSS for windows (v.15.01) program .Analysis of variance (ANOVA) was performed on all data from each batch and treatment of goats’ and ewes’ milk cheeses at different ripening stages. Comparisons of mean values of physicochemical, microbiological and BA were followed by Duncan test with significance level set on 10 CFU/g, probably because strains of Lactococcus lactis were added as the starter culture, while lactobacilli counts were significantly lower at the beginning of the ripening, although their counts rose as the ripening progressed, achieving similar counts to lactococci. Enterococci counts remained steady at a level about 6 log10 CFU/g at the beginning of the process, showing a slight decrease around the 15th and the 30th day in the goats’ and ewes’ cheeses, respectively. S. aureus, enterobacteria and E. coli counts decreased during the ripening until becoming undetectable in most of the samples. These results are in agreement with those reported by other authors in other varieties of cured cheeses [In control samples (non HHP-treated) of both types of cheese, Lactoccocci was the main microbial group at the beginning of the ripening, showing counts above 8 log cheeses ,28,29,3010 CFU/g and 1.5 log10 CFU/g in the goats’ and ewes’ milk cheeses, respectively. In the case of lactobacilli mean reductions were 3.41 log10 CFU/g and 1.03 log10 CFU/g. However, these counts recovered along the ripening process and no statistical differences were observed at the end of the work with respect to control samples. Ewes’ milk cheeses HHP15 showed a reduction of about 1.91 log10 CFU/g and 1.33 log10 CFU/g for lactoccocci and lactobacilli counts, respectively. In that case the subsequent recovery of these counts was not so clear and they did not achieve the same counts than control samples. Novella-Rodríguez et al. [10 CFU/g in goats’ milk cheeses as a consequence of HHP, although a subsequent increase was found during ripening. Rynne et al. [10 CFU/g in starter and non-starter LAB in cheddar cheese treated at 400 MPa on the first day of ripening. In ovine milk ripened cheeses treated on day 1 and 15 at 300 MPa and 400 MPa proved to cause similar reductions although recovery of LAB counts was observed only in samples treated 1st day. HHP treatments also affected significantly (p < 0.05) the counts of enteroccocci in both type of cheeses in either HHP1 and HHP15 samples, being unable to recover the initial counts in any case. Arqués et al. [10 CFU/g in enteroccocci counts when a treatment of 400 MPa for 10 min at 10 °C was applied on the 2nd day of ripening to “La Serena” cheeses, remaining constant during the rest of the ripening. Although high counts of enteroccoci have been associated with the unhygienic processing of cheese, their presence is also considered important for the development of the typical aroma and flavour of traditional Mediterranean cheeses. Their counts may range from 104 to 106 CFU/g in curds and 105 to 107 CFU/g in ripened cheeses [S. aureus HHP1 treatments also caused significant reductions in both kind of cheeses and HHP15 in ewes’ milk cheeses, becoming undetectable in most cases at the last day of the work. Nevertheless, the initial counts were already quite low. Similar results were reported in “La Serena” cheeses treated at 400 MPa on the 2nd day of ripening [S. aureus has been described as one of the most HHP resistant non-sporulated bacteria. López-Pedemonte et al. [10 CFU/g.HHP treatments reduced significantly the Lactic Acid Bacteria (LAB) counts. In HHP1 treated samples, lactoccocci counts decreased 2.2 logz et al.  also obse et al.  reporteds et al.  reportedripening  and in Cripening . S. auree et al.  describe10 CFU/g of enterobacteria in ewes’ milk cheeses after a 400 MPa treatment applied the 1st and the 15th day of ripening. Initial counts of E. coli were significantly lower than enterobacteria. O’Reilly et al. [E. coli to HHP in cheeses with reductions above 6 log10 CFU/g after HHP treatments at 400 MPa.HHP treatments showed to be very effective reducing Gram-negative bacteria except for HHP1 in goats’ milk cheese samples, where a slight growth was noted on the 15th day probably due to a possible recovery of the sub lethal injured cells after the HHP treatment. However, cheese ripening conditions, with low pH, increasing salt concentration and presence of LAB, made difficult this recovery to consolidate and no positive counts were detected later at the 30th and 45th days of ripening. Juan et al.  also desy et al. , Capellay et al.  and De Ly et al.  have preThe proteolytic activity parameters measured in the goats’ and ewes’ cheeses are presented in Significant differences between control and HHP1 samples in TNBS were observed from the 45th to the 60th days of ripening in goats’ milk cheeses, while the differences in FAA content were mainly noted from day 30 to 60. In both parameters control samples showed the highest values. The ratio of RI displayed a different trend where an increment of around two times was observed during the first 15 days period in control and HHP-treated samples but after this point the proteolysis rate became slower. In the ewes' milk cheeses, an increment on the three-proteolysis index evaluated was observed during the ripening period . In the WSN is produced mainly by the rennet and to a lesser extent by plasmin or cellular proteinases, whereas starter peptidases are primarily responsible for the formation of small peptides and free amino acids [RI values indicated that proteolysis was more intense during the first 15 days of ripening in the goats’ and ewes’ cheeses in control and HHP treated samples. This proteolytic activity was probably caused by milk and rennet proteinases, being not so clear the role of microbial proteinases. On fact, no acids . Messensno acids  observedno acids  in a stuno acids  found inWSN/TN values than control samples, at the end of the ripening but higher than those obtained in samples with HHP-treatment on the 1st day. In contrast, some works reported after application of HHP treatments not differences on proteolysis indexes during ripening in Gouda [On ewes’ milk cheeses HHP15 samples showed slightly higher values on the three-proteolysis index evaluated than control samples during the ripening period, although no significant differences were observed at the end of the ripening, reflecting that this treatment did not significantly affect the proteolysis process. However, the HHP application during the initial stages of the ripening in ovine and caprine milk cheeses led to a decrease of the proteolysis rate. Similar results were obtained by Juan et al.  who in pin Gouda  and chedin Gouda  or an inin Gouda ,42. Starin Gouda , being oin Gouda . HHP-indin Gouda  and possin Gouda  indicatein Gouda  pointed in Gouda  and Calzin Gouda  noticed in Gouda ,42 suggeEnterococcus spp. and LAB isolated from cheese samples [TY and PU were the main BA formed in untreated goats’ milk cheeses, showing concentrations of about 492 and 476 mg/kg, respectively, at the end of the ripening. Whereas in ewes’ milk control samples the predominant BA were TY and CA with 277 and 106 mg/kg, respectively. Several authors reported, in variable ranges, TY (88.6–445 mg/kg), HIS (not detected–697 mg/kg), PU 74.15–446.5 mg/kg) and CA (44–269.77 mg/kg) as the most abundant BA in goats’ and ewes’ milk ripened cheeses .5 mg/kg ,55,56. T samples ,59,60,61Enterococcus spp. also have shown this capability “in vitro” [Lactococcus lactis, are also able to form PU via the agmatine deiminase [PU amounts in control goats’ milk cheeses were almost the same than TY but HHP1-treatments caused a decrease on the first 15 days, remaining almost stable throughout the rest of the ripening. PU levels were about 83% lower in the HHP1 samples than in control samples at the 60th day. The pressure application in ewes’ milk cheeses also affected the PU amounts formed, showing that HHP1 treatments limited the production of this diamine around 93% compared with control cheeses at the end of the ripening. HHP15 showed to be less efficient reducing the formation of PU. With respect to CA, untreated and HHP-treated goats’ milk cheeses displayed amounts below 100 mg/kg without appreciate significant differences between them. In control ewes’ milk cheeses this diamine increased mainly during the first 15 days, while in HHPI and HHP15 cheeses remained without significant changes throughout the ripening. The amounts of TR and PHE increased during the ripening in control goats’ milk cheese samples without significant differences in relation to HHP1-treated samples, while low amounts were detected in ewes’ cheeses remaining practically constant during ripening and without showing significant differences between treatments. Formation of PU and CA is usually associated with Gram negative bacteria, although some strains of n vitro” . Some LAeiminase . The appeiminase .Enterobacteriaceae strains able to decarboxylate histidine in diverse foodstuffs [In goats’ raw milk control cheeses the concentration of HIS was very low at the beginning of ripening but increased later reaching its maximum after 45 days (18 mg/kg). No significant changes were observed until the 60th day. In HHP1-treated samples HIS showed a similar behaviour but in this case, differences were found after the 45th day displaying levels 68% lower than control samples at the 60th day of ripening. HHP1 ewes’ milk cheese samples showed a reduction of 92% of HIS when compared with control samples. This treatment was more efficient than the HHP15. Diverse authors reported low HIS amounts in cheese (below 100 mg/kg), relating the production of this BA with some LAB ,56,63,64odstuffs ,67,68,69odstuffs ,59,70.In cheeses made from raw goat milk, low levels of polyamines were found at the beginning of ripening, increasing their concentration very slightly during ripening until the 60th day . HoweverThe effectiveness of HHP treatments depends on different factors, such as the type of cheese, the stage of ripening, the HHP processing conditions applied, the kind and number of microorganisms present. In this work, the use of HHP applied at the initial phases of ripening affected significantly the microorganisms responsible of forming BA, and, in consequence, reduced its content, especially of TY and PU in goats’ milk cheeses and TY and CA in ewes’ milk cheeses, assuring the safety of this product for the most BA sensitive consumers. As previously mentioned, HIS and TY are the BA that most frequently have been related with food-borne outbreaks, being suggested threshold values in cheese between 200–400 mg/kg of HIS ,72,73 an


Secukinumab has been approved for the treatment of moderate to severe plaque psoriasis. However, safety measures concerning drug administration is vital during treatment. Understanding the right way to administer drugs is important to reduce any serious adverse drug event. In this analysis we aimed to systematically show the risk of adverse drug events which were observed with 150 mg versus (vs) 300 mg secukinumab for the treatment of moderate to severe plaque psoriasis.www.ClinicalTrials.com and EMBASE were searched for relevant publications based on the comparison of secukinumab 150 mg vs 300 mg for the treatment of moderate to severe plaque psoriasis. Adverse drug events were considered as the clinical endpoints. Statistical analysis was carried out by the RevMan 5.3 software. Risk ratios (RR) and 95% confidence intervals (CIs) were generated to represent the data following statistical analysis.The major online databases: Cochrane Central, MEDLINE, P = .94), the risk of serious adverse events  and the risk of adverse events leading to drug discontinuation  were not significantly different between 150 mg vs 300 mg secukinumab for the treatment of moderate to severe plaque psoriasis. When the detailed adverse drug events were studied, the risks of infection or infestation , naso-pharyngitis , headache , diarrhea , pruritus , arthralgia , upper respiratory tract infection , hypertension , nausea , and cough  were still not significantly different between these 2 dosage regimens.Seven studies with a total number of 2361 participants were included. Results of this analysis showed that the risk of any adverse event (RR: 1.00, 95% CI: 0.96–1.05; Secukinumab 150 mg and 300 mg were both equally tolerable and might safely be used for the treatment of moderate to severe plaque psoriasis. No significant adverse drug events were observed with any of the dosage. There are several types of psoriasis, however, plaque psoriasis is the most common comprising of up to 90% of cases.Psoriasis which is an autoimmune disorder of the skin, has reached more than seven million cases in the United States.Plaque psoriasis is characterized by raised red patches covered with a whitish build-up of dead skin and it mainly appears on the elbow, knees, scalp, and lower back, but it might also be present in the other surfaces of the body.To understand the treatment of psoriasis, it is necessary to have a knowledge of the pathogenesis involved in the development of this chronic disease. In brief, the mechanism of this autoimmune disease is based on the expression of the cytokine interleukin 17A. This novel drug was approved by the Food and Drug Administration in January 2015 for the treatment of adults with moderate to severe plaque psoriasis and was prescribed as secukinumab 150 mg and 300 mgrespectively.Secukinumab (Cosentyx), a human IgG1k monoclonal antibody, binds and inhibits/neutralizes interleukin 17A and this is how it is involved in the treatment of psoriasis. However, the dosage to be used was often a question to both the physicians and the patients.Several recent research showed secukinumab to be effective in the treatment of moderate to severe plaque psoriasis. Drugs might be harmful even if they are meant to improve the health of a patient and taking them correctly and understanding the right way to administer them is important to reduce any serious adverse drug event.Safety measures concerning drug administration is vital during the treatment of patients with specific drugs.In this analysis we aimed to systematically show the risk of adverse drug events which were observed with 150 mg versus (vs) 300 mg secukinumab for the treatment of moderate to severe plaque psoriasis.22.1www.ClinicalTrials.com and EMBASE were searched for relevant publications based on the comparison of secukinumab 150 mg vs 300 mg for the treatment of moderate to severe plaque psoriasis. The time frame for retrieval of articles was from the beginning of August to the end of September 2018.The major online databases: Cochrane Central, MEDLINE, Only English publications were considered for this research article and the search process was carried out using the following search terms:Secukinumab and psoriasis;Secukinumab and plaque psoriasis;Secukinumab and moderate to severe psoriasis;Secukinumab, psoriasis and adverse drug events.2.2Inclusion criteria was based on studies which:Were randomized trials or observational studies;Compared secukinumab 150 mg vs 300 mg for the treatment of moderate to severe plaque psoriasis;Reported adverse drug events as their clinical endpoints;Reported data which could be used in this meta-analysis.Exclusion criteria was based on studies which:There were not randomized trials and observational cohorts;Did not compare secukinumab 150 vs 300 mg for the treatment of psoriasis;Did not report the relevant adverse drug events;Reported data which were not compatible with this analysis;Were duplicated studies.2.3Most of the participants were patients who were treated for moderate to severe plaque psoriasis as shown in Table The adverse drug events which were assessed included:1.Any adverse event;2.Serious adverse events;3.Adverse events leading to drug discontinuation;4.Infection or infestation;5.Naso-pharyngitis;6.Headache;7.Pruritus;8.Arthralgia;9.Upper respiratory tract infection10.Hypertension;11.Nausea;12.Cough.Four studies reported a follow-up time period of 52 weeks, 2 studies reported a follow-up time period of 12 weeks and one study reported a follow-up time period of 16 weeks as shown in Table 2.4The original studies were reviewed and the following data were extracted by the respective four authors :1.Total number of participants assigned to 150 vs 300 mg secukinumab respectively;2.The endpoints reported, with respective follow-up time periods;3.The baseline characteristics of the participants;4.The main features of the original articles;5.The total number of adverse events in each category of subgroup. whereby bias risk was labelled as ‘low risk’, ‘moderate risk’ and ‘high risk’.The methodological quality of the trials was assessed with reference to the recommendations from the Cochrane collaborationAny disagreement which followed was completely resolved by consensus.Statistical analysis was carried out by the RevMan 5.3 software. Risk ratios (RR) and 95% confidence intervals (CIs) were generated to represent the data following statistical analysis.P value less or equal to .05 was considered as statistically significant, and a result reporting a P value above .05 was considered statistically insignificant.Heterogeneity was assessed by the Q statistic test whereby a result with a 2 statistic test. The I2 value was represented by percentage. A higher percentage of I2 denoted a higher heterogeneity whereas a lower percentage denoted a lower heterogeneity.Heterogeneity was also assessed by the I2 value was less than 50%, whereas a random statistical model was used if the I2 value was more than 50%.In addition, a fixed statistical model was used if the ISensitivity analysis was carried out by a method of exclusion. Each study was excluded at a time and a new analysis was generated to observe for any significant difference from the main results.Publication bias was visually observed by assessing funnel plots.2.5Ethical approval or board review approval was not required for this study.33.1 a total number of 675 publications were retrieved from online databases. Following a careful assessment of the titles and abstracts, 596 articles were eliminated due to irrelevance.Following the PRISMA guideline,Seventy-nine (79) full text articles were assessed for eligibility.Following further assessments, other full text publications were eliminated due to the following reasons:Literature review (2);Meta-analysis and pooled studies (7);Did not report relevant endpoints (6);Control group was absent (7);Did not report the relevant dosage of drug (2);Included data which could not be used (3);Duplicated studies (45).–16 were included in this meta-analysis. The flow diagram for the study selection has been demonstrated in Figure Finally, only 7 studies3.2The main features of the original studies were listed in Table A total number of 2361 participants were included in this meta-analysis comparing 150 mg vs 300 mg secukinumab for the treatment of moderate to severe plaque psoriasis. The 1169 participants were assigned to 150 mg secukinumab whereas 1192 participants were assigned to 300 mg secukinumab as shown in Table Six studies were randomized trials and 1 study was an observational cohort.The enrollment time period of the participants varied from year 2011 to 2015.2. A minor population of the participants also suffered from psoriatic arthritis. Duration of psoriasis varied from 7.5 to 20.4 years as shown in Table The baseline characteristics of the participants were listed in Table 3.3P = .94), the risk of serious adverse events  and the risk of adverse events leading to drug discontinuation  were not significantly different between 150 mg vs 300 mg secukinumab for the treatment of moderate to severe plaque psoriasis as shown in Figure Results of this analysis showed that the risk of any adverse event , naso-pharyngitis , headache , diarrhea , pruritus , arthralgia , upper respiratory tract infection , hypertension , nausea , and cough  were still not significantly different between 150 mg vs 300 mg secukinumab for the treatment of moderate to severe plaque psoriasis as shown in Figures When the adverse drug events were studied in details, the risks of infection or infestation  criteria for authorship for this article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published.LZ, HY, QC and JZ were responsible for the conception and design, acquisition of data, analysis and interpretation of data, drafting the initial manuscript and revising it critically for important intellectual content.Conceptualization: Li Zhang, Hua Yang, Qihong Chen, Jing Zhao.Data curation: Li Zhang, Hua Yang, Qihong Chen, Jing Zhao.Formal analysis: Li Zhang, Hua Yang, Qihong Chen, Jing Zhao.Funding acquisition: Li Zhang, Hua Yang, Qihong Chen, Jing Zhao.Investigation: Li Zhang, Hua Yang, Qihong Chen, Jing Zhao.Methodology: Li Zhang, Hua Yang, Qihong Chen, Jing Zhao.Project administration: Li Zhang, Hua Yang, Qihong Chen, Jing Zhao.Resources: Li Zhang, Hua Yang, Qihong Chen, Jing Zhao.Software: Li Zhang, Hua Yang, Qihong Chen, Jing Zhao.Supervision: Li Zhang, Hua Yang, Qihong Chen, Jing Zhao.Validation: Li Zhang, Hua Yang, Qihong Chen, Jing Zhao.Visualization: Li Zhang, Hua Yang, Qihong Chen, Jing Zhao.Writing – original draft: Li Zhang, Hua Yang.Writing – review & editing: Li Zhang, Hua Yang.


The considerable microbial diversity of soils and key role in biogeochemical cycling have led to growing interest in their global distribution and the impact that environmental change might have at the regional level. In the broadest study of Arctic soil bacterial communities to date, we used high-throughput DNA sequencing to investigate the bacterial diversity from 200 independent Arctic soil samples from 43 sites. We quantified the impact of spatial and environmental factors on bacterial community structure using variation partitioning analysis, illustrating a nonrandom distribution across the region. pH was confirmed as the key environmental driver structuring Arctic soil bacterial communities, while total organic carbon (TOC), moisture and conductivity were shown to have little effect. Specialist taxa were more abundant in acidic and alkaline soils while generalist taxa were more abundant in acidoneutral soils. Of the 48 147 bacterial taxa, a core microbiome composed of only 13 taxa that were ubiquitously distributed and present within 95% of samples was identified, illustrating the high potential for endemism in the region. Overall, our results demonstrate the importance of spatial and edaphic factors on the structure of Arctic soil bacterial communities. Arctic soil bacterial communities are influenced by spatial and edaphic factors and exhibit biogeographical distribution. Despite the extent, it should be noted that large areas of Canada and Russia remain uncharacterized and under-represented, highlighting areas where sampling should be prioritized in the future. Approximately 150 g of soil per sample was collected from the top 15 cm, avoiding plant roots and rocks and using an ethanol-cleaned shovel and Whirl-Pak bags . Remaining plant roots and rocks were removed manually in a class II microbiological safety cabinet; samples were homogenized by manual mixing and frozen at −20°C before transportation to the United Kingdom. Samples were conserved at –20°C until analyzed.Soil samples were collected across the Arctic region between April 2017 and September 2017, and the global positioning system (GPS) coordinates of each site were recorded with a portable GPS  was measured gravimetrically by heating previously dried soils to 550°C for 4 h. pH and conductivity were measured in the laboratory in a 1:5 freshly thawed soil to water ratio, using a Mettler-Toledo FE20 pH meter  and a CMD500 conductivity meter .et al. et al. Soil DNA was extracted for each sample using the PowerSoil kit , for a total of 400 DNA extracts. Each sample was amplified by polymerase chain reaction (PCR) using the universal primers 515F-806R, as per the Schloss lab standard operating procedure  calling was performed on high-quality trimmed sequences at 97% similarity level using the USEARCH  in QIIME  and significant dbMEM vectors.Variation partitioning analysis (VPA) was used to assess the impact of environmental and spatial factors on Bray–Curtis dissimilarity. The linear trend was considered to represent a source of variation like any other and therefore, we performed variation partitioning analysis using the environmental variables, Generalist and specialist taxa were identified by selecting taxa present in over 95% of samples within each unique and overlapped pH category. Indicator taxa were determined by the Dufrene-Legendre indicator species analysis using the multipatt function  and Planctomycetes (15%). Acidobacteria represented 13% while Actinobacteria, Bacteroidetes, Chloroflexi and Verrucomicrobia each accounted for 4–6% of the community. Of these 48 147 OTUs, only 135 had overall abundances over 0.1% across all 386 samples and were defined as abundant taxa. They accounted for 32% of all the reads, illustrating the dominance of a few taxa over the rest of the community. Within these abundant taxa, Acidobacteria dominated the subcommunity at ∼32% with Blastocatellia (12.8%) and Acidobacteria subgroup 6 (7.2%) as abundant classes. Alphaproteobacteria (10.6%) and Betaproteobacteria (6.6%) were the most commonly identified Proteobacteria  and Verrucomicrobia was largely represented by Spartobacteria (17.2%). Actinobacteria (10.7%), Chloroflexi (7%) and Bacteroidetes (3.5%) were also among the abundant phyla classified.R2 = 0.108, P < 2.2 × 10−16). The best-fitted power model  suggested the presence of spatial autocorrelation, also qualified as dispersal limitation, within the first 500 m. For a more accurate estimate of dispersal limitation, we plotted the same graph within 0 and 500 m  were produced; after forward selection, two significant MEMs were identified for the total community and used in further analyses. The variation partitioning analysis differentiated the effect of environmental factors, linear trend and spatial vectors on Bray–Curtis dissimilarity  on ity Fig. . Resultsity Fig. , confirmOf the environmental variables we measured, we identified pH as the primary factor structuring Arctic soil bacterial communities. The Pearson correlations indicated that pH did not have strong collinearity (coefficient over |0.8|) with any other variable measured . The envWe compared the observed richness and Shannon diversity index by pH range Fig.  and B usIn terms of community composition, we differentiated the abundant community, composed of only 135 OTUs, from the total community composed of 48 147 OTUs and observed the changes in relative abundance by pH range. In the total community, Acidobacteria, Armatimonadetes, Planctomycetes and Verrucomicrobia decreased with increasing pH. On the other hand, Bacteroidetes, Chloroflexi and Gemmatimonadetes increased in alkaline soils; Proteobacteria remained stable throughout. In the abundant community, Acidobacteria, Bacteroidetes and Gemmatimonadetes increased with increasing pH while Actinobacteria, Planctomycetes and Verrucomicrobia remained stable Fig.   .The differentiation of specialist from generalist taxa associated with pH range was conducted by considering all taxa identified in this study and present in a minimum 95% of all samples from each pH category Fig. . 125 aciWe conducted the indicator species analysis on the abundant taxa only as they may be more useful for monitoring purposes. We identified 17 unique taxa-habitat associations. In acidic soils, 10 indicator species, mainly Acidobacteria group 1 and 2 and Ca. Methylacidiphilum were identified. The six OTUs associated with Acidoneutral soils were mainly Acidobacteria group 4 (Blastocatellia) and Spartobacteria (Verrucomicrobia). Finally, only one OTU was identified as an indicator species for alkaline soils and belonged to the Holophagae (Acidobacteria). We also conducted the indicator species analysis of abundant taxa to combine pH ranges and identified 84 OTUs. 21 were identified as indicator species of acidic and acidoneutral soils combined, and mainly belonged to the Verrucomicrobia. Only two OTUs were associated with acidic and alkaline soils, Acidothermus (Actinobacteria) and Candidatus Xiphinematobacter (Verrucomicrobia), further illustrating the low overlap of taxa between these ecosystems and inferring the large ecosystem differences. Finally, 61 OTUs were associated with acidoneutral and alkaline soils, mainly belonging to the Acidobacteria and Proteobacteria phyla  in bacterial community structure and diversity while TOC, moisture and conductivity explained between 2 and 5% of the variance, respectively. The identification of pH as a key factor influencing Arctic soil bacterial community composition is in line with previous Arctic studies over both, small and large scales  and stochastic  processes. It has been hypothesized that communities primarily structured by deterministic processes will host more specialist taxa, highly adapted to the ecosystem, while communities influenced by dispersal will harbor primarily generalist taxa, more resilient to change  could illustrate the need for environmental adaptations to survive in these ecosystems and suggests that deterministic processes likely structure microbial communities. Geographically, the first cluster identified by the Bray–Curtis dissimilarity matrix Fig.  grouped et al. et al. 4 emissions come primarily from wetlands, the identification of these phylotypes in acidic soils suggests wetland may not be the only major source of northern methane emissions. Understanding the distribution and abundance of such indicator taxa, combined with field gas measurements, may allow large-scale estimates of climate-active gases release rates in the region, from soils and wetlands (Wartiainen et al. et al. The indicator species analysis determined OTU-pH range associations identifying different classes of Acidobacteria and Verrucomicrobia, primarily, characteristic of each pH range. The identification of abundant indicator species with strong habitat associations opens the possibility of predicting the presence and abundance of these taxa across the Arctic region. This is especially important for taxa such as Ca. Methylacidiphilum, a known methanotroph unlike others as it belongs to the Verrucomicrobia phylum instead of the Proteobacteria (Dunfield This study investigated patterns in soil bacterial diversity across the Arctic and demonstrated that bacterial communities differed across the region. However, the dispersal range was limited to ∼20 m and the variation in communities across the Arctic was primarily driven by environmental rather than spatial factors. The key edaphic property influencing communities we identified was pH, in accordance with that seen elsewhere. However, 63% of the variation remained unexplained, calling for more in-depth investigations of the drivers of bacterial community structure in the region. We identified unique and indicator taxa closely associated to the soil pH. Finally, a core microbiome composed of only 13 ubiquitously distributed taxa was identified, highlighting the potential for endemism in the region. While this study brings a deeper understanding of Arctic bacterial community assemblages, this is also a baseline for future functional studies in the region, which will be critical to forecast the ecological consequences of environmental change.fiz128_Supplemental_FilesClick here for additional data file.


Stentor coeruleus. 16S rRNA gene metabarcoding was performed on single cells of S. coeruleus and on their environment, water from the sewage stream. Our results showed that the prokaryotic community composition differed significantly between Stentor cells and their environment. The core microbiome common for all ciliate specimens analyzed could be defined, and it was composed mainly by representatives of bacterial genera which include also potential human pathogens and commensals, such as Neisseria, Streptococcus, Capnocytophaga, Porphyromonas. Numerous 16S rRNA gene contigs belonged to endosymbiont “Candidatus Megaira polyxenophila”. Our data suggest that each ciliate cell can be considered as an ecological microniche harboring diverse prokaryotic organisms. Possible benefits for persistence and transmission in nature for bacteria associated with protists are discussed. Our results support the hypothesis that ciliates attract potentially pathogenic bacteria and play the role of natural reservoirs for them.Microbiomes of multicellular organisms are one of the hottest topics in microbiology and physiology, while only few studies addressed bacterial communities associated with protists. Protists are widespread in all environments and can be colonized by plethora of different bacteria, including also human pathogens. The aim of this study was to characterize the prokaryotic community associated with the sessile ciliate  Protists may also host associated bacteria, but their microbiomes still are not investigated, and ciliates seem to be perfect candidates for this purpose. They are relatively big and bacterivorous, a number of representatives of this abundant phylum are easily recognizable and can be maintained in cultures. Ciliates have been intensively studied in wastewaters, focusing on their role as indicators of process efficiency5 and as detectors of heavy metal pollution7. Indeed, ciliates as filter-feeders are also efficient removers of some pathogens11, and contribute, together with disinfection systems, in pulling down the microbial load13, thus improving the quality of the effluent discharged by wastewater treatment plants14. At the same time, bacteria may benefit in finding protection inside the host cell from disinfection systems and chemical substances used to reduce bacterial load15. The phagotrophic activity of ciliates allows natural entrance of bacteria into the eukaryotic host, thus enabling the establishment of symbiotic associations with bacteria, also occasionally pathogenic ones16. Indeed, the ciliate cell offers a great variety of intracellular compartments suitable for bacterial colonization, and symbiotic bacteria have been described in the ciliates’ cytoplasm, nuclear apparatus, mitochondria and even in perinuclear space17.All possible forms of coexistence of prokaryotes with metazoan organisms became one of the most rapidly developing research fields in microbiology, and also in physiologyRickettsiaceae20 or Francisellaceae22. Symbiotic associations can be classified according to the stability of bacteria and ciliate interaction in three categories: permanent, highly infectious, and accidental17. The first two groups comprise all associations, which were recorded more than once in literature, and have been studied from ecological and evolutionary points of view. However, accidental invaders are never considered as true symbionts, such bacteria are likely poorly adapted for symbiotic persistence, and cannot be maintained for a long time within the host cell17. Such invaders usually have been reported just once17, and the study of these temporary occasional associations with “classical” microbiological and molecular techniques is rather difficult, as the associations are very unstable and promptly get lost. Several studies reported that in ciliates the establishment of symbiotic associations with pathogenic bacteria can be experimentally induced23. For example, ciliates under certain conditions may host Legionella pneumophila25, that sometimes leads to increase of virulence of these bacteria for human cells26. Internalized Listeria remain infectious in cysts of Tetrahymena27, and pathogenic Escherichia coli survives after passage through Tetrahymena cells28. However, such systems do not appear to be stable. The role of protists as environmental reservoirs of pathogenic bacteria has been elucidated more in amoebae, in which several pathogens have been found30, for example Francisella tularensis32, Vibrio cholerae33, E. coli29, Chlamydia34, Mycobacterium37, and Listeria monocytogenes38. Thus, in some cases, pathogenic bacteria also are a part of prokaryotic communities associated to protists, but for most of them the mode of maintenance and propagation in environment remains unclear.Ciliates often host endosymbionts phylogenetically related to pathogenic bacteria belonging to the families Euplotes focardii has been reported39. It was shown that even during long maintenance of ciliates in laboratory conditions they still keep a rather variegated set of initial bacterial cohabitants. Recently, Illumina sequencing was applied to discriminate between bacteria of the rumen fluid and symbiotic prokaryotes of ruminant ciliates40. The results of the pilot study showing that marine and freshwater ciliates harbour distinct microbial communities were reported in the latest publication41, and such prokaryotic communities were analyzed for several Paramecium samples42. However, up to now just two latter works remain the only studies of prokaryotic communities associated with single ciliate cells by NGS. This gap in knowledge extends also to other groups of protists, as there were only few attempts to assess the microbiomes of free-living amoebae44 applying NGS, and in several works bacterial diversity in association with protists was estimated by cloning and sequencing49. Also, few reports analyzed microbial consortia associated to cyanobacteria52 proving that even bacteria may organize and maintain stable communities of cohabiting prokaryotes. Nevertheless, all these few studies assume that unicellular organisms do have their own microbiomes.However, contribution of bacteria in survival and environmental adaptation of protists, as well as the role of protists as reservoirs for the pools of microorganisms remains poorly understood. The studies of protistan microbiomes just start to be addressed with the diffusion of new technologies, such as Next Generation Sequencing (NGS). The characterization of the bacterial consortium associated with the Antarctic marine ciliate Stentor coeruleus , followed by Cyanobacteria (19.3%), Proteobacteria (14.6%), Actinobacteria (11.7%), Bacteroidetes (1.9%), Planctomycetes (1.7%), Chloroflexi (1.3%), and Euryarcheota (1.2%). On the contrary, the most abundant phylum in Stentor cells was Proteobacteria (66.7%), followed by less copious Bacteroidetes (15.6%), Firmicutes (6.9%), Cyanobacteria (5.3%), Fusobacteria (2.7%), and Actinobacteria (1.0%).In total representatives of 11 phyla were found, and among them 8 were abundant in all environment samples, while 6 phyla were the most plentiful in lls Fig. . BacteriRhizobiales (uncultured MNG7), uncultured Actinobacteria (bacterium PeM15), uncultured Cyanobacteria, uncultured Clostridiales, and some Firmicutes. Some potential pathogens and opportunistic bacteria like Streptococcus sp. were also detected in the environmental samples. Only few OTUs found in the environments were rather frequent also in the ciliate samples, though showing different average percentages , was identified as the chloroplast sequence of the microalga Mychonastes homosphaera (Chlorophyceae), which once has been found in symbiotic association with Stentor polymorphus55. In the same Stentor cell, OTU belonging to Holospora obtusa, a specific bacterial endosymbiont of the ciliate Paramecium caudatum, was found, but its abundance was very low (0.20%) , human commensals , potential pathogens or bacteria related to pathogens , and symbionts (i.e. known as obligate intracellular bacteria) Table . The setNeisseriaceae and to the genus Neisseria, normally associated to human mucosa, were recorded in association with stentors, and never in the environmental controls. Interestingly, Streptococcus sp. appeared to be mostly associated to Stentor cells, despite its presence was detected also in the environment. The non-parametric Kruskal-Wallis test assessed that there was a significant difference in the distribution of OTUs between stentors and their environments (p < 0.05), thus showing that human commensals and potentially pathogenic bacteria were indeed preferentially associated to Stentor cells. However, the most abundant OTU in six of seven Stentor samples belonged to the widespread endosymbiotic bacterium “Candidatus Megaira polyxenophila” , probably inhabiting Stentor cells  remained the most abundant phylum, it was followed by Proteobacteria (15.8%), Actinobacteria (14.0%), Bacteroidetes (2.6%), Planctomycetes (2.2%), Chloroflexi (1.7%), and Cyanobacteria (1.7%). Removal of “Ca. Megaira polyxenophila” reads significantly reduced the fraction of Proteobacteria in the ciliates samples (32.0%), while the most abundant phyla associated with ciliates became Bacteroidetes (38.5%), then followed by Firmicutes (16.4%), Fusobacteria (6.3%), Actinobacteria (2.3%), and “Ca. Saccharibacteria” (2.1%), while Cyanobacteria (0.6%) became almost absent , accordingly with the results of NMDS.Several analyses were carried out to test if the prokaryotic community composition of the environments and of the Stentor cells grouped together, and were separated from the environments , or previously found in activated sludge (uncultured bacteria from “Ca. Saccharibacteria”63) , and uncultured Rhizobiales MNG7, probably inhabiting the stream. Majority of environmental bacteria were absent or negligible in the communities associated with Stentor cells.Prokaryotic community disclosed in the environmental samples was diverse and especially rich with several bacterial phyla, first of all ) Tables , 2 were S. coeruleus cells isolated from the neighboring locations of the sewage stream were characterized by rather diverse associated bacterial communities, which included several dozens of different representatives , bacterial intranuclear symbiont strictly host-specific for another ciliate, Paramecium caudatum, were detected in the dataset of the same Stentor cell  against SILVA database as “uncultured Rickettsiaceae”.Unexpectedly, few reads belonging to Stentor samples  are a part of human microbiome73. Majority of them are known to colonize the mucosal surfaces of mammals, and are considered as opportunistic bacteria, if not true pathogens, which may cause serious diseases under certain conditions60. The interaction between Streptococcus sp. and Veillonella sp. was studied during the formation of biofilms74, which could be interesting since they have been both found in association with Stentor cells and might have a role in bacterial aggregation. The genus Neisseria and other representatives of the family Neisseriaceae were found in rather high abundancy , or to the groups of commensals (Leptotrichiaceae). We suggest to consider such bacteria as transitory components of Stentor microbiome, which by chance manage to colonize some ciliate cells.Some free-living bacteria, like a representative of me Table . NumerouStentor microbiome. These bacteria are very likely prone to interact with eukaryots, as they belong to genera encompassing numerous commensals or even pathogens of animals. When they appear freely in water, they may use their general skills to interact with available eukaryotic cells, namely, protists. There are two possible ways of bacterial persistence in association with protists: to stay attached on the surface of the host cell, or to resist digestion or even to escape from the food vacuoles to the host cytoplasm. Ciliates are rather big protists . The sampling points were located about 10 m downstream one after another, starting with PW1.Each collected sample (200 ml) was divided in two subsamples: 150 ml were immediately filtered through 0.2 μm nitrocellulose filters  for further NGS analysis of the environmental controls, and 50 ml were brought to the laboratory within 15 min for isolation of ciliates. The filters were air-dried and stored at −80 °C till DNA extraction.Stentor cells were observed in vivo, and identified as S. coeruleus .The filters were firstly treated by sonication as describedStentor samples were centrifuged for 30 min at 14000 rpm and 4 °C, and the pellet was dried for 3–5 minutes in the same PCR tube. Then 15 μl of MilliQ water and a mixture of glass beads (0.1 and 0.5 mm in diameter) were added to the tubes in approximate ratio 3:1. The mixture was homogenized with Tissue Lyser LT  for 3 minutes at a maximal frequency of 50 Hz. The suspension was vortexed and centrifuged at 14000 rpm for 10 min. The lysate was transferred to a new tube, the glass beads were washed in 15 μl of MilliQ water, centrifuged again, and the supernatant was transferred in the tube with the lysate.Just before DNA extraction, the 88. Two of nine Stentor samples did not yield sufficient DNA for library preparation and were discarded. The DNA libraries were sequenced on the MiSeq platform .Preparation of the DNA libraries was performed according to the Illumina protocol  using primers for V3 and V4 region of the 16S rRNA genePreparation of the DNA libraries and sequencing was carried out in the Center of Shared Scientific Equipment “Persistence of microorganisms” at the Institute for Cellular and Intracellular Symbiosis, UrB RAS .89. De-multiplexing and quality filtering were performed removing any low quality or ambiguous reads, and sequences shorter than 200 nucleotides were discarded. Chimeras were identified using UCHIME90 and subsequently removed from the analysis. Operational Taxonomic Units (OTUs) were clustered with a 97% of similarity cutoff using USEARCH91, and SILVA 119 database92 as reference. The most common sequence was selected in each OTU as representative. Taxonomic classification up to genus level was performed using Ribosomal Database Project Classifier (RDP)93 against the SILVA 119 database.Raw FASTQ files were analyzed using the Quantitative Insights Into Microbial Ecology 1.9.1 software package (QIIME)Stentor cell samples, and the community richness was evaluated calculating the Shannon index. The significance of OTUs presence in the ciliates samples was assessed using the non-parametric Kruskal-Wallis test. Furthermore, similarities between stentors and their environments were assessed using the non-metric Multi-Dimensional Scaling (nmMDS) and Principal Coordinate Analysis (PCoA) based on the unweighted Unifrac distance matrices. In addition, beta diversity was estimated to compare the bacterial communities of the Stentor cells and their environments. To confirm the significance of differences observed between bacterial communities of the environments and stentors, ANOSIM was calculated. The same analyses were carried out also after excluding the most abundant OTUs representing symbiotic microorganisms associated with stentors (see Results).QIIME was used to evaluate diversity of the prokaryotic communities both within and between samples. Firstly, the relative abundances of prokaryotic taxa were estimated as the percentage of contigs number for each taxon for the environmental and the Dataset 1


This study was carried out to isolate and screen actinomycetes from soil of two salterns in Taza-Morocco, for the production of antimicrobial compounds against a set of target bacteria. Also, it aims to highlight some practices in order to isolates actinomycetes and screen for their ability to produce antibacterial compounds.Soil samples were analyzed for physical and chemical parameters including pH, electrical conductivity, and salinity. The actinomycetes were isolated on Casein Starch Agar (CSA) medium and purified on International Streptomyces Project 2 (ISP-2) medium. Antimicrobial activity of actinomycete isolates was evaluated by measuring the inhibition zone. These activities were tested against Dickeya solani IP2222, Pectobacterium brasiliensis 13471a, Escherichia coli K12, Proteus mirabilis, Pseudomonas aeruginosa CECT118, Listeria innocua CECT4030, Staphylococcus aureus CECT976, Bacillus subtilis DSM 347 and Candida alibicans, using three different culture media  and at two temperatures of incubation (30°C and 37°C).Physical and chemical analysis of soil samples showed that both sites are alkaline. Also, with regards to salinity, the second site showed to contain high salt concentration compared the first site. The abundance of bacteria isolated on CSA medium from both sites showed correlation with the physical-chemical properties of the sampling soils. Incubation temperature of 30°C resulted in a high number of actinomycetes (18/22) isolates with antimicrobial effect relative to the temperature of 37°C (4/22). Some actinomycetes isolates show antimicrobial effect on only one culture medium, which shows a special nutritional requirement to express their antimicrobial effect. On the other hand, some isolates, they express their antimicrobial effect on the three media at the same time. Additionally, some isolates of actinomycetes inhibit the growth of several microorganisms at once. While others inhibit the growth of only one microorganism tested which reflects a possible specificity of antimicrobial substances.Growth conditions including, media composition, temperature of incubation and the spectrum of test strain tailors the behavior of the antimicrobial screening. They are widely distributed in nature, particularly in soil . They co CFU/ml) . Microor CFU/ml) . The culStudied sites: the sampling sites in this study were soil of a saltern  and pH of soil samples. CONSORT-Model C535 -G. BOYER-S/N 71658 was used in this regard. For samples preparation for pH determination, 10g of soil sample were mixed with 50ml of distilled water. After agitating for 15 minutes, the suspension was allowed to settle for 2 h [ for 2 h . For theMedia: the Casein Starch Agar (CSA) medium used for actinomycetes isolation contained  was used for isolation of actinomycetes [omycetes . This meomycetes . The antomycetes  and MuelStrains: the strains tested (s tested  were culIsolation of actinomycetes: 10 g of each soil sample were suspended in 100 ml of sterile physiologic water (0.9% NaCl in distilled water). Heat treatment was performed in a water bath at 50°C for 60 min under agitation [gitation . Serial Purification and conservation of actinomycetes isolates: actinomycetes isolates were recognized by their morphological aspects and by microscopic observation. Isolates were purified by successive streaking on ISP2 medium. They were stored in 20% glycerol at -80°C and/or maintained at 4°C for less than 1 month.Antimicrobial activity of actinomycetes isolates: antimicrobial activities of pure isolates were determined using the double layer agar method test according to the protocol described by Ouhdouch [+8 - 4.10+8 CFU/ml) and incubated for 48 h at 30°C or at 37°C. Antimicrobial activity was evaluated by measuring the inhibition zone. Each test was carried out in triplicate. For the negative control, the tested strains were grown in the same culture medium without inoculation with actinomycetes.Ouhdouch . Pure acSoil chemical analysis: pH values obtained for two sites are greater than 7, which qualifies our biotope in the category of calcareous soils . Also, the results obtained show that the second site is very salty compared to the first site, which is qualified salty  were obtained on CSA medium from the two sites, 13 isolates from the first site and 9 from the second site. In site 1, green isolates are dominant. However, in site 2, white isolates are dominant  ( (≈ 82%) . On the  (≈ 82%) .Effect of culture medium on antimicrobial substances production: screening results of actinomycetes isolates incubated at 30°C producing antimicrobial substances showed that from the 22 isolates, 11 (50%) are capable of producing antimicrobial substances on Bennett medium, 10 (> 45%) on CSA medium and only 3 on MH medium (> 13%) ( (> 13%) , Table 5Listeria innocua on CSA medium and inhibits Candida albicans on Bennett medium, isolate S12 inhibits Proteus mirabilis on CSA medium and inhibits Dickeya solani on Bennett medium and isolate S18 inhibits Proteus mirabilis on CSA medium, inhibits Listeria innocua on Bennett medium and inhibits Pectobacterium brasiliensis on Mueller Hinton medium.Actinomycetes are known as versatile producers of antimicrobials active metabolites . ScreeniDickeya solani IP2222, Escherichia coli K12 and Proteus mirabilis) and 2 isolates (9%) against Gram-positive bacteria (Staphylococcus aureus CECT976 and Listeria innocua CECT4030). In relation to incubation temperature, 37°C negatively affects the growth of actinomycetes isolates and consequently diminishing the capacity of production of antimicrobial substances. In 2001, Ouhdouch and collaborators reported that Bennett's medium achieved a maximum number of antifungal activities. Also, production tests of antifungal substances at different temperatures , show that the temperature of 30°C allows a better production of antifungal substances [Other studies have reported that the Gelatin Broth (GB) medium ) is favorable to the production of antimicrobial agents in contrary to ISP2 medium . ScreeniActinomycetes are the most prolific of all microorganisms as producers of antibiotics. Exploitation of particularly extreme new biotopes is recommended to isolate new species of actinomycetes of biotechnological interest. This is the first study carried out on isolation of actinomycetes from soils of saline salterns conducted in Taza region, Morocco. Control of growing conditions and nutritional requirements of these isolates are very important to find new natural substances with valuable biological activities.Actinomycetes are the main source of the vast majority of bioactive secondary metabolites;Actinomycetes are produced many antibiotics, that are best recognized and most valuable. These antibiotics include amphotericin, nystatin, chloramphenicol, gentamycin, erythromycin, vancomycin, tetracycline, novobiocin, neomycin, etc;The search for new bioactive molecules from actinomycetes remains a priority objective to make the rapid development of microbial resistance to multiple drugs and their undesirable side effects.The isolation of actinomycetes from unexplored ecosystems is one of the most attractive sources for the search for new bioactive metabolites;The production of secondary metabolites by actinomycetes depends on the choice of incubation temperature, the use of suitable culture media and the nature of the target pathogen.The authors declare no competing interests.