Lactobacillus crispatus, Lactobacillus iners, and Gardnerella vaginalis on the cervical epithelial barrier function and determined whether L. crispatus mitigates the effects of lipopolysaccharide (LPS) and G. vaginalis on the cervical epithelial barrier as a possible mechanism by which CV microbiota mitigates disease risk. Ectocervical and endocervical cells treated with L. crispatus, L. iners, and G. vaginalis bacteria-free supernatants alone or combined were used to measure cell permeability, adherens junction proteins, inflammatory mediators, and miRNAs. Ectocervical and endocervical permeability increased after L. iners and G. vaginalis exposure. Soluble epithelial cadherin increased after exposure to L. iners but not G. vaginalis or L. crispatus. A Luminex cytokine/chemokine panel revealed increased proinflammatory mediators in all three bacteria-free supernatants with L. iners and G. vaginalis having more diverse inflammatory effects. L. iners and G. vaginalis altered the expression of cervical-, microbial-, and inflammatory-associated miRNAs. L. crispatus mitigated the LPS or G. vaginalis-induced disruption of the cervical epithelial barrier and reversed the G. vaginalis-mediated increase in miRNA expression. G. vaginalis colonization of the CV space of a pregnant C57/B6 mouse resulted in 100% PTB. These findings demonstrate that L. iners and G. vaginalis alter the cervical epithelial barrier by regulating adherens junction proteins, cervical immune responses, and miRNA expressions. These results provide evidence that L. crispatus confers protection to the cervical epithelial barrier by mitigating LPS- or G. vaginalis-induced miRNAs associated with cervical remodeling, inflammation, and PTB. This study provides further evidence that the CV microbiota plays a role in cervical function by altering the cervical epithelial barrier and initiating PTB. Thus, targeting the CV microbiota and/or its effects on the cervical epithelium may be a potential therapeutic strategy to prevent PTB.Cervicovaginal (CV) microbiota is associated with vaginal health and disease in non-pregnant women. Recent studies in pregnant women suggest that specific CV microbes are associated with preterm birth (PTB). While the associations between CV microbiota and adverse outcomes have been demonstrated, the mechanisms regulating the associations remain unclear. As the CV space contains an epithelial barrier, we postulate that CV microbiota can alter the epithelial barrier function. We investigated the biological, molecular, and epigenetic effects of  Preterm birth (PTB), defined as delivery at less than 37 weeks of gestation, remains the leading cause of perinatal morbidity and mortality. Of all live births reported in 2016, 9.8% were preterm deliveries. Babies born prematurely often face, not only neonatal, but also, lifelong medical complications leading to an estimated 26.2 billion dollars in annual medical and societal expenses in the United States alone . DespiteSpontaneous PTB (sPTB) is well recognized to be a complex syndrome as it has been associated with multiple disease mechanisms including inflammation and infection, stress, uteroplacental ischemia, and uterine overdistension among many others . The mosCervical remodeling is a natural continually occurring process over the course of gestation that is broken down loosely into four phases: softening, ripening, dilation, and postpartum repair . A normaLactobacillus bacterial species are common inhabitants of the human vagina and are typically  indicative of a healthy vaginal space due to their production of lactic acid (and resulting low pH) (Lactobacillus spp. including L. crispatus (CST I), L. gasseri (CST II), L. iners (CST III), and L. jensenii (CST V), the other two CSTs (CST IV-A and IV-B) are composed of mostly anaerobic bacteria, Gardnerella vaginalis , Mobiluncus, Atopobium vaginae, Bacteroides, and Prevotella. These microbes are typically associated with unhealthy or dysbiotic vaginal states such as bacterial vaginosis (BV), HIV, and adverse obstetrical outcomes, including sPTB  , recent  low pH) . In heal low pH) . While fing sPTB . The conand sPTB . Howeverand sPTB . Traditiand sPTB . In thesof women . In anotof women . HoweverL. crispatus, from CST I, which is the most predominant bacteria in the human vagina and is normally considered a representative of a healthy CV space  CV space ; (2) L. ysbiosis , HIV, anysbiosis ; and (3) with BV . For thi2 humidified incubator.Ectocervical  (Ecto) and endocervical  (Endo) cell lines  were cultured in keratinocyte serum-free medium (K-SFM) supplemented with 0.1 ng/ml epidermal growth factor and 50 μg/ml bovine pituitary extract , 100 U/mL penicillin, and 100 μg/mL of streptomycin at 37°C in a 5% COLactobacillus crispatus (L. crispatus) is a clinical human isolate that was collected, sequenced, identified, and gifted to us by Jacques Ravel at the University of Maryland. Lactobacillus iners  and Gardnerella vaginalis  (Table 1. L. crispatus was grown in NYCIII media with 10% horse serum at 37°C in an anaerobic jar in a 5% anaerobic CO2 incubator for 7 days. L. iners was grown in Tryptic Soy Broth (TSB)  with 5% defibrillated sheep blood  at 37°C in an anaerobic jar in a 5% anaerobic CO2 incubator for 7 days. G. vaginalis was grown in Tryptic Soy Broth with 5% defibrillated rabbit blood  at 37°C in an anaerobic jar in a 5% anaerobic CO2 incubator for 7 days. The cultures were centrifuged two times for 10 min each at 2,500 rpm at 4°C to remove the bacteria. The resulting supernatants were sterile-filtered through a 0.22 μM membrane filter  to remove any remaining bacterial components or debris. The bacteria-free supernatants and each bacterium’s specific growth media (negative control to determine the baseline measurements of background growth media) were then used for in vitro cell culture experiments.C 14018)  were purSupplementary Table 1).The consistency of bacterial growth from week to week (and between experiments) was assessed by consistent optical density readings in the 450–600 nm range. Additionally, colony-forming unit assays (CFUs) were performed after 72 h of growth to determine the amount of bacteria per milliliter from which the bacteria-free supernatants were collected  bacteria-free supernatants in K-SFM cell growth media. For cells treated with bacteria-free supernatants from L. crispatus, the K-SFM medium was supplemented with 50 mM HEPES and sodium bicarbonate  to bring the pH of the medium up to a physiological level (7.2) as L. crispatus bacteria produces high amounts of lactic acid during growth. Without pH adjustments, even at lower volume per volume percentages, the cells did not survive. Ectocervical cells were treated with L. crispatus (n = 3), L. iners (n = 3), and G. vaginalis (n = 3) bacteria-free supernatants for 48 h. In additional experiments, ectocervical cells were dually treated with bacteria-free supernatants of L. crispatus and G. vaginalis where the cells were exposed to L. crispatus supernatants (5% v/v) on day 1 followed by G. vaginalis supernatants (5% v/v) on day 2 or vice versa for 24–48 h (n = 6–9). For all experiments, cells were also treated with the L. crispatus, L. iners, or G. vaginalis bacterial growth media alone as a negative control to determine any baseline effects of the growth media on the outcome of interest. At the end of each experiment, cell culture media were collected for ELISA assays and/or the cells were collected in TRIzol for RNA extraction.Ectocervical cells were plated at 2.0 × 106 cells/ml into 24-well hanging cell culture inserts, which contain 1 μm pores with a transparent polyethylene terephthalate (PET) membrane precoated with collagen. The cells were treated with 10% (v/v) bacteria-free supernatants from L. crispatus (n = 6), L. iners (n = 6), and G. vaginalis (n = 9) for 48 h. In additional experiments, the ectocervical cells were treated with the three bacteria-free supernatants as described above and 24 h later, the cells were exposed to the bacterial by-product, lipopolysaccharide   for additional 24 h (n = 6–9). In a third set of experiments, ectocervical cells were dually treated with supernatants from L. crispatus and G. vaginalis (as described above) for 24–48 h (n = 9). For all experiments, cells were also treated with the L. crispatus, L. iners, or G. vaginalis bacterial growth media alone as a negative control to determine any baseline effects of the growth media on cell permeability. After 72 h of total growth time, the media were removed and phenol red-free K-SFM media  containing FITC-Dextran was added to the top of the insert. The movement of FITC-Dextran from the top insert to the bottom was measured after 2 h by using a fluorescent plate reader at 485 and 535 nm, excitation and emission, respectively.Ectocervical and endocervical cell permeability was determined using an In Vitro Vascular Permeability Assay kit . Briefly, ectocervical and endocervical cells were plated at 1.0 × 10L. crispatus, L. iners, and G. vaginalis alone. The levels of sECAD, IL-6, and IL-8 were measured by ligand-specific commercially available ELISA kits that use a quantitative sandwich enzyme immunoassay technique using regents from R&D Systems .Ectocervical cells were cultured in six-well plates and treated with bacteria-free supernatants as stated above. Soluble E-cadherin (sECAD), IL-6, and IL-8 were measured in ectocervical cell culture media after 48 h of treatment with bacteria-free supernatants or bacteria growth media from L. crispatus (n = 3), L. iners (n = 3), and G. vaginalis (n = 3) bacteria-free supernatants. The samples were run in duplicate, per the manufacturer’s protocol on the FLEXMAP3D Luminex platform . Data were analyzed using a standard curve generated by a five-parameter logistic (5PL) curve fit using Xponent 4.2 software (Luminex).Ectocervical cells were cultured and treated with bacteria-free supernatants as stated above. A 41-plex human cytokine/chemokine magnetic bead Luminex panel I  was run on ectocervical cell culture media after 48 h of treatment with TM, Rockland, DE, United States) prior to the generation of cDNA.Following the bacteria-free supernatant treatments in six-well plates described above, ectocervical cells were washed in sterile PBS and collected in TRIzol  and underwent phenol–chloroform extraction. The resulting aqueous phase was further column-purified with the miRNeasy kit  according to the manufacturer’s protocol for total RNA isolation including small RNAs. RNA concentration was determined via a NanoDrop 2000 Spectrophotometer  (Qiagen) for SYBR Green. qPCR was performed on the QuantStudio 7 Flex Real-Time PCR System  using the miScript SYBR Green PCR kit (Qiagen) according to the manufacturers’ protocol. The ΔΔCT method was used for relative expression quantification using the QuantStudio Real-time PCR software v1.1 . The endogenous reference gene RNU6B was used for miRNA quantification. All miRNA primer sets were purchased from Qiagen: miR-143 (MS00003514), miR-145 (MS00003528), miR-193b (MS00031549), miR-146a (MS00003535), miR-223 (MS00003871), miR-148b (MS00031458), miR-15a (MS00003178), miR-21(MS00009079), miR-142 (MS00031451), miR-494 (MS00033754), miR-30e (MS00009401), and RNU6B (MS0001400).C57/B6 timed-pregnant mice were purchased from Charles River Laboratories . We considered E0 as mating day and E1 was determined based on the presence of copulatory plug. Animals were shipped on day 10 after mating and housed individually in our facilities. These animals were acclimated for 4 days before experiments were performed. All the experiments were performed in accordance with the National Institutes of Health Guidelines on Laboratory Animals and with approval from the University of Pennsylvania’s Institutional Animal Care and Use Committee (IACUC #:805513).G. vaginalis colonization was created in the following manner. Timed-pregnant, C57/B6 mice, were anesthetized with isoflurane on embryonic day 14 (E14) and five CV lavages were performed with 100 μL of sterile PBS prior to control treatment or bacterial inoculation. Bacterial doses were determined using published data in a non-pregnant mouse model  PTB outcome and (2) CV colonization by G. vaginalis. For the first trial, on E14, E15, and E16, the animals received an intravaginal inoculation of G. vaginalis (n = 9) by inserting a sterile pipette tip and injecting 20 μL of 5 × 109 CFU/animal or sugar water  (n = 6) into the mouse vagina. These time points were chosen to mimic a change in the CV microflora at mid-pregnancy. Immediately post-inoculation, each animal was positioned in dorsal decubitus under isoflurane anesthesia for 3 min and 100% pure petroleum jelly  was added to the cervix with a sterile swab to ensure that the inoculum would remain within the CV space. The animals were observed twice daily for PTB until E19 (term). In the second animal trial, on E14 the animals received an intravaginal inoculation of G. vaginalis (n = 5) of 5 × 109 CFU/animal or sugar water (n = 4). Twenty-four hours later, the animals were sacrificed and CV fluid (CVF) was collected to assess for G. vaginalis colonization of the CV space. The CVF was collected by gently rinsing the CV space (pipetting in and out seven times) with 100 μL of sterile PBS. The washes were pooled together into one sterile tube for each dam.A pregnant mouse model of se model , and theG. vaginalis gDNA, we used a TaqMan 16S specific probe to this bacterium . gDNA from the CVF was quantified by QPCR to determine tissue-specific colonization. A standard curve was created from serially diluted gDNA from G. vaginalis to quantify the amplification. This standard curve was used for relative quantification of G. vaginalis abundance using the 7900HT Real-Time PCR System (Applied Biosystems). The results were analyzed using the RQ manager software v2.4 (Applied Biosystems). In TaqMan QPCR assays, the relative abundance of the target of interest was divided by the relative abundance of pan-bacterial 16S rRNA in each sample to generate a standardized abundance for the target transcript of interest.Genomic DNA (gDNA) was isolated and purified from the CVF with the ZR fecal MiniPrep DNA extraction kit . To quantify the amount of p < 0.05), then pair-wise comparison with a Tukey post hoc test was performed for data that had similar variances. However, if the variances between groups were significantly different, then a Sidak post hoc test was performed. If data were not normally distributed, then the non-parametric Kruskal–Wallis test was used and pairwise comparison was performed using a Dunn’s multiple comparison test. To compare two groups , unpaired t-tests were performed. If the data did not have equal variances, then an unpaired t-test with Welch’s correction was done.Statistical analyses were performed for all experiments with the GraphPad Prism Software . For data that were normally distributed, one-way analysis of variance (ANOVA) was performed. If statistical significance was reached  was unchanged after treatment with L. crispatus supernatants (p = 0.8660), while L. iners (p < 0.0001), and G. vaginalis (p < 0.0001) supernatants significantly increased cell permeability. A similar effect was seen in endocervical cells , as cell permeability was unchanged in the presence of bacteria-free supernatants from L. crispatus (p = 0.2942) and increased by L. iners (p = 0.0006), and G. vaginalis (p < 0.0001). There was no significant effect of the bacterial growth media alone on cell permeability of ectocervical or endocervical cells for any of the bacteria tested. Additionally, the increase in cell permeability after treatment with bacteria-free supernatants from both L. iners (p = 0.0002) and G. vaginalis (p < 0.0001) in ectocervical cells and G. vaginalis (p < 0.0001) only in endocervical cells was significantly higher than the bacterial growth media alone.To determine if common vaginal bacterial species release factors within the CV space that have the ability to alter the cervical epithelial barrier, we performed cell permeability assays in ectocervical and endocervical cells after treatment with bacteria-free supernatants. Ectocervical cell permeability  and G. vaginalis (p < 0.0001) supernatants significantly increased sECAD concentration when compared to non-treated cells (control) . sECAD concentration was unchanged by both L. crispatus and L. iners growth media; however, there was a significant increase in sECAD after exposure to G. vaginalis growth media (p < 0.0001). Therefore, the elevated sECAD concentration measured after exposure to G. vaginalis bacteria-free supernatants was due to an effect of the growth media and not the secreted factors produced by the bacteria. sECAD was significantly higher after exposure to L. iners bacterial supernatant when compared to L. iners growth media alone (p = 0.0009) while G. vaginalis bacteria-free supernatant versus G. vaginalis growth media were not significantly different.As we observed similar significant changes in cell permeability with both ectocervical and endocervical cells, we chose to use ectocervical cells as a model system in all of the following experiments that were performed to further elucidate the mechanisms regulating the observed changes in cervical epithelial barrier function. We sought to investigate if bacteria-free supernatants were able to alter the adherens junction complex that plays a critical role in the epithelial cell-to-cell adhesion needed to maintain the cervical epithelial barrier. As we have previously shown that cleavage of E-cadherin can act as a biomarker for reduced adherens junction and cervical epithelial barrier integrity , we measL. crispatus, L. iners, and G. vaginalis bacteria-free supernatants. Of the 41 cytokines/chemokines present on the Luminex assay, 21 were found to be expressed by ectocervical cells . A list of those cytokines/chemokines included in the Luminex assay that were not detectable in either non-treated (control) nor bacteria-free supernatant-treated ectocervical cells can be found in Supplementary Table 2. Overall, the results of the Luminex assay revealed a significantly varied and diverse immune profile between non-treated (control) ectocervical cells and those exposed to bacteria-free supernatants. After setting an arbitrary cutoff and focusing only on those cytokines/chemokines exhibiting greater than a five-fold change with a significant p-value <0.05, there were five cytokines/chemokines increased by L. crispatus, eight cytokines/chemokines increased by L. iners, and eight cytokines/chemokines increased by G. vaginalis . Assuming that these cytokines/chemokines are acting in their traditional roles, out of the three bacteria-free supernatants tested, L. crispatus was the only bacterial supernatant to significantly increase the classical anti-inflammatory cytokine, IL-10 (p < 0.0001) .As we have previously shown that the activation of an inflammatory response by the bacterial by-product LPS can initiate cervical epithelial breakdown , we wantFigures 3B,C, p < 0.001) after treatment with bacteria-free supernatants from L. crispatus, L. iners, and G. vaginalis. IL-6 and IL-8 concentrations were also significantly upregulated by L. crispatus, L. iners, and G. vaginalis growth media (p < 0.001). IL-6 and IL-8 levels were significantly decreased after exposure to L. crispatus bacteria-free supernatants when compared to its growth media (p < 0.01). On the contrary, both cytokines were increased after exposure to G. vaginalis bacteria supernatants versus G. vaginalis growth media (p < 0.0001). For L. iners, IL-6 expression was significantly increased after bacteria-free supernatant exposure when compared to growth media alone (p < 0.0001); however, IL-8 expression was unchanged between the two groups. Therefore, the elevated IL-6 and IL-8 concentration measured after exposure to L. iners bacteria-free supernatants was due to an effect of the growth media and not the secreted factors produced by the bacteria.To validate the results of the Luminex assay and to assess any background results due to the bacterial growth media alone, we measured the classical proinflammatory cytokines known to be highly produced by ectocervical and endocervical cells , IL-6 anL. crispatus, L. iners, and G. vaginalis bacteria-free supernatants have the ability to alter specific miRNAs in ectocervical cells. Eleven miRNAs were chosen for further investigation based on previously published studies showing an association with sPTB, shorter gestation, and breakdown of the cervical epithelial barrier . L. crispatus bacteria-free supernatants had no effect on the expression of any of the miRNAs measured . With the exception of miR-223 , no significant changes in miRNA expression were seen with L. crispatus, L. iners, or G. vaginalis growth media alone. miRNAs showing no significant changes in expression after exposure to any of the three bacteria-free supernatants tested are listed in Supplementary Table 3.As alterations in miRNA expression have been shown to play a role in cervical epithelial cell function , we asse barrier . EctocerE. coli-activated immune response. Previous studies conducted by our laboratory have shown that LPS has the ability to initiate PTB in an animal model , as we have shown previously (p < 0.0001 vs. control). In the presence of L. crispatus bacteria-free supernatants, the LPS-induced increase in cell permeability was significantly inhibited . However, in the presence of both L. iners  and G. vaginalis  bacteria-free supernatants, the LPS-induced increase in cell permeability was significantly enhanced. Following exposure to L. crispatus, L. iners, and G. vaginalis bacteria growth media containing LPS, ectocervical cell permeability was not significantly different than with LPS alone.As we have shown that bacteria-free supernatants have the ability to alter cervical cell permeability, we wanted to determine if these supernatants could augment or mitigate cervical cell permeability in the presence of a general gram-negative al model  and thatal model . Therefoeviously , LPS aloL. crispatus has a protective effect against inflammation-mediated cell permeability, we investigated if L. crispatus bacteria-free supernatants have the ability to mitigate the increased cell permeability observed in the presence of G. vaginalis. Exposure of ectocervical cells to L. crispatus bacteria-free supernatants followed by G. vaginalis bacteria-free supernatants resulted in a significant reduction in the G. vaginalis-induced increase in cell permeability . Additionally, exposure of ectocervical cells to G. vaginalis bacteria-free supernatants first followed by a secondary exposure to L. crispatus bacteria-free supernatants resulted in a similar significant reduction in the G. vaginalis-induced increase in cell permeability . The dual exposure to L. crispatus and G. vaginalis bacterial growth media had no effect on cell permeability independent of the order of exposure.As L. crispatus bacteria-free supernatants against the increases in cell permeability seen with the G. vaginalis bacterial supernatants, we investigated if a reduction in the G. vaginalis-induced miRNA expression could be a potential mechanism. We investigated the effects of the dual exposure to L. crispatus and G. vaginalis bacteria-free supernatants in ectocervical cells on the expression of only those miRNAs that were found to be increased after G. vaginalis treatment including miR-143, miR-145, miR-193b, miR-146, miR-223, miR-148b, and miR-15a . G. vaginalis bacteria-free supernatant-induced increases in miR-143, miR-146, miR-223, miR-148b, and miR-15a were all significantly reduced in the presence of L. crispatus bacteria-free supernatant independent of the order of supernatant exposure . G. vaginalis bacteria-free supernatant induced increases in miR-145  and miR-193b  were only significantly reduced when the ectocervical cells were exposed to L. crispatus first followed by G. vaginalis but not the opposite. L. crispatus and G. vaginalis growth media alone or in combination had no effect on miRNA expression for any of the miRNAs measured.Given the protective effects of G. vaginalis bacteria-free supernatants are able to significantly alter cervical epithelial cell function, including the cellular processes regulating cervical remodeling, we wanted to investigate if the presence of G. vaginalis in the CV space of pregnant mice would result in preterm delivery. Intravaginal inoculation of G. vaginalis into the CV space resulted in a 100% PTB rate over a 96 h time period after initial exposure on E14 . The sugar water control animals had no preterm deliveries over the same time period. To provide evidence that the intravaginal G. vaginalis inoculations resulted in the colonization of the CV space, we collected CVF, isolated gDNA, and measured G. vaginalis 16S rRNA 24 h after the first exposure. G. vaginalis 16s rRNA was significantly increased in the CVF of mice when compared to the sugar water controls .As we have shown that L. crispatus, L. iners, and G. vaginalis have the ability to regulate the cervical epithelial barrier through multiple diverse mechanisms including cleavage of adherens junction proteins, altered inflammatory profiles, and differential epigenetic regulation through elevated miRNA expression. Additionally, this study shows that L. crispatus, a bacterium often associated with a “healthy” CV space, has the ability to protect the cervical epithelial barrier in the presence of either a general CV infection (LPS) or in the presence of bacteria associated with increased risk of BV, STI/STDs, or HIV . Interestingly, the protection conferred by L. crispatus bacteria-free supernatants in the presence of G. vaginalis seems to be mediated, at least in part, through miRNA-dependent mechanisms. Additionally, we show that G. vaginalis inoculated into the CV space of mice results in PTB, which suggests that the cervical dysfunction caused by G. vaginalis secreted factors are mechanistically linked to the cervical remodeling process. The results of this study support our hypothesis that the secreted factors derived from the CV microbiome play a significant role in regulating cervical epithelial cell function and that a dysbiotic state within the CV space could result in a disruption of the cervical epithelial barrier. The data from this study begin to ascribe the mechanisms linking secreted by-products from a specific CV microbiota to a direct cervical function – maintenance of the cervical epithelial barrier – and has implications for women’s health including not only adverse obstetrical outcomes (PTB) but also the acquisition of sexually transmitted diseases .This study provides novel evidence that bacteria common to the CV space play a significant role in regulating cervical epithelial cell function. We have shown that supernatants derived from G. vaginalis (BV) or L. iners  may have the ability to degrade the epithelial mucous layer resulting in biofilm production, cytolysis and inflammation of the cervical epithelial barrier   and MMP-8 concentrations in human cervical vaginal fluid  plays a significant role in both non-pregnant and pregnant states where it predominately acts to prevent the invasion of microbial pathogens. However, recent studies have suggested that the presence of known vaginal microbial pathogens such as  barrier . The bree cervix . Similars (MMPs) . The relal fluid , providiL. crispatus, L. iners, and G. vaginalis elicited a widely varied and complex immune response. Perhaps not surprisingly and in agreement with previous studies  show no difference between L. iners bacteria-free supernatants and the background control media indicating that L. iners, a bacteria with intermediate effects on CV health   . The ambesponse) . TherefoL. crispatus, L. iners, and G. vaginalis. Interestingly, the upregulation of miR-143, miR-145, miR-146, miR-223, miR-148, and miR-15a by G. vaginalis bacteria-free supernatants and miR-193b, miR-146, and miR-223 by L. iners bacteria-free supernatants with no expression change in any of the measured miRNAs by L. crispatus shows the presence of a clear bacterial species-specific alteration in miRNA expression profiles. As one miRNA is known to regulate hundreds of downstream targets, it is of interest to investigate these miRNAs in a cluster as there will certainly be redundancy in their functions. As a cluster, these miRNAs have been previously associated with cervical epithelial barrier breakdown , but not commensal (L. crispatus), have the ability to epigenetically alter cervical cell function. Follow-up studies with in-depth investigation into each of these miRNAs and their downstream targets are warranted to identify the specific biological pathways regulating the effect of these miRNAs on cervical cell function. Additionally, future studies would be needed to ascertain the long-term effects of L. iners and G. vaginalis on the epithelial cell function within the CV space, as this study only investigated miRNA expression at one time point during bacteria-free supernatant exposure (48 h post-exposure). Taken together, these data provide evidence of a role for secreted factors derived from the CV microbiome in the induction of epigenetic modification of cervical epithelial cells in the presence of a dysbiotic state.The disruption of the cervical epithelial barrier has been linked to both inflammation-dependent and independent mechanisms, as we have previously shown that an upregulation of PTB-associated miRNAs, miR-143, and miR-145 in the cervix has the ability to breakdown the cervical epithelial barrier due to diverse mechanisms, including the regulation of cell adhesion, apoptosis, and cell proliferation . Multiplreakdown , PTB  or bacteria associated with an increased risk of CV disease . The results of this study show that L. crispatus protects the cervical epithelial barrier in the presence of an inflammatory stimulus; however, the mechanisms regulating this interaction will need further investigation as these experiments are not able to identify if L. crispatus bacteria-free supernatants have the ability to block LPS binding to the TLR4 receptor or activate the host immune response to prevent the inflammatory insult. While there are previous studies that have shown a protective effect of L. crispatus in the face of Prevotella bivia and G. vaginalis (BV-associated bacteria)  thus reducing the infection rate  to the vaginal mucosa  , E. coli or fetal death in utero . Since hormone levels (estrogen and progesterone) are already elevated in the pregnant mouse, exogenous administration of hormones was not required in our model, unlike the non-pregnant models. The creation of a pregnant mouse model with intravaginal G. vaginalis colonization will provide a novel system in which to further study the effects of G. vaginalis on both vaginal and cervical epithelial dysfunction as well as the interactions between bacterial and host immunity. We acknowledge that the colonization of the CV space with a single bacterial species does not represent the human CV microbial community as a whole and limits the study of the interaction between bacterial species on host cervical function. However, this animal model does provide clear evidence of a biological mechanism linking a specific bacterium, G. vaginalis, to PTB.While our CV space , this stCD1 mice . Lower lin utero . Previouaginalis , Group Balactiae , and Canalbicans . SimilarL. crispatus, L. iners, and G. vaginalis, play a significant role in regulating cervical epithelial cell function in both normal and dysbiotic states. Secreted factors produced by bacteria associated with increased risk of disease , L. iners and G. vaginalis, disrupt the cervical epithelial barrier by diverse mechanisms including the cleavage of adherens junction proteins, the upregulation of proinflammatory immune mediators, and increased miRNA expression. G. vaginalis colonization within the CV space of mice resulted in PTB providing further evidence for a role of G. vaginalis in cervical epithelial cell dysfunction, which leads to a disruption of the cervical epithelial barrier and the initiation of cervical remodeling. Protection of the cervical epithelial barrier conferred by L. crispatus secreted factors in the presence of infection-inducing bacteria  provides evidence for a beneficial effect of L. crispatus to women who may be at a higher risk for PTB. Future studies are warranted to investigate if therapeutics that promote an L. crispatus dominate CV state would be useful in the prevention of STI acquisition and/or PTB by preventing cervical epithelial dysfunction.Overall, the results from this study show that bacteria common to the CV space, LA wrote the manuscript, created the figures, and analyzed the data. LA, AB, and ME conceived and designed the experiments. LA, AD, L-JS, GB, and LH performed the experiments. All authors contributed to manuscript revision and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gossypium spp.) has been maintained for the last four decades in Multan, Pakistan. This geographical location has been observed as a hotspot for the evolution of begomoviruses and satellites associated with cotton leaf curl disease (CLCuD). Recent studies showed that begomoviruses responsible for the CLCuD epidemic in the 1990s, and that almost disappeared from the CLCuD complex in 2000s, have been observed again in CLCuD-infected cotton fields. To identify host species that acted as probable reservoirs for these viruses, we characterized begomoviruses and satellites in non-cultivated cotton species G. raimondii, G. thurberi and G. mustelinum and identified several species of CLCuD associated begomoviruses and satellites. Further, phylogenetic analysis indicated that the identified begomoviruses and beta/alphasatellites are closely related to the ones associated with the most recent CLCuD complex. qPCR indicated that the comparative level of virus significantly decreased in the presence of alphasatellites. Our results indicated that non-cultivated cotton species have been continuously challenged by diverse begomoviruses and associated satellites and act as reservoirs for CLCuD associated begomoviruses. These results provide novel insights into understanding the spread of begomoviruses and associated satellites in New World cotton species introduced into the Old World.A collection of cultivated and non-cultivated species of cotton ( Begomovirus (family Geminiviridae) of plant viruses, that are transmitted via the insect vector, whitefly (Bemisia tabaci) [Cotton is the world’s largest fiber producing crop and determines the economy of major producers like Pakistan and India . However tabaci) .Begomoviruses are classified into bipartite and monopartite; bipartite having two genomic components DNA-A and DNA-B, and monopartite having a single genomic component similar to the DNA-A of bipartite begomoviruses. DNA-A (genome size ~2.7 kb) of begomoviruses encodes the replication associated protein (Rep), replication enhancer protein (REn), transcriptional activator protein (TrAP), coat protein (CP), V2 and C4 proteins required for virus replication and gene expression. DNA-B (genome size ~2.7 kb) encodes a movement protein (MP) and nuclear shuttle protein (NSP) required for virus inter- and intra-cellular movement .begomovirus. Alpha Rep has also been reported to be a suppressor of post-transcriptional gene silencing (PTGS) [Begomoviruses in the Old World (OW) are usually associated with ~1.4 kb subgenomic betasatellites, which determine the pathogenicity and symptom severity of the virus. Betasatellites encode a single βC1 protein and depend on their helper virus for their replication, encapsidation and movement . Alphasag (PTGS) ,7. Some g (PTGS) ,9. So fag (PTGS) .Gossypium genus belongs to the family Malvaceae and includes 46 diploid, five true tetraploid and one purported tetraploid species [Gossypium species originated from a common ancestor and diversified into eight groups, from A-G and K [G. arboreum like A genome and G. raimondii like D genome [G. arboreum has been cultivated for fiber production in the Indian subcontinent for hundreds of years [G. hirsutum was introduced to the Indian subcontinent in 1818 from Mexico and because of its high production, it covers 98% of the cultivated land area where G. arboreum was previously grown [G. arboreum, G. hirsutum was not resistant to the co-evolved plant pathogens such as CLCuD causing begomoviruses [The  species ,12. All -G and K . All tetD genome . Diploidof years . Tetraplly grown . Howeveroviruses .begomovirus species, cotton leaf curl Kokhran virus (CLCuKoV), cotton leaf curl Multan virus (CLCuMuV), cotton leaf curl Rajasthan virus (CLCuRaV), cotton leaf curl Alabad virus (CLCuAlV), tomato leaf curl Bangalore virus (ToLCBaV) and papaya leaf curl virus (PaLCuV) [G. arboreum remained symptomless and presented immunity to CLCuD where G. hirsutum, on the other hand, remained highly susceptible [G. arboreum as a result of grafting with CLCuD infected G. hirsutum, but it is still resistant under field conditions [CLCuD was first observed in the 1960s, and the first epidemic emerged in 1986 in the Multan area of the Punjab province in Pakistan . In the (PaLCuV) ,21. As f(PaLCuV) . The cha(PaLCuV) . Several(PaLCuV) . However(PaLCuV) . This be(PaLCuV) ,27,28. Dceptible . There anditions . In Pakistan, a collection of non-cultivated and cultivated cotton species have been maintained for the last four decades at the Central Cotton Research Institute in Multan . Non-cultivated cotton species are being maintained for breeding purposes as they could be a potential source of genetic resistance against biotic and abiotic stresses. This geographical location and the surrounding areas have been the center of CLCuD and vector whitefly infestation since the first epidemic of CLCuD in Pakistan in the 1990s. There is a frequent exchange of whiteflies among cultivated and non-cultivated cotton species, allowing virus transmission and maintenance in the non-cultivated species. Due to the distinct genotypes and perennial growth habit of non-cultivated species, they supposedly impose different selection pressure on the viruses they harbor as compared to annually grown cultivated cotton. Thus, a unique virus population is maintained in these plants via whitefly transmission within this herbarium . We haveG. raimondii (diploid D5), G. thurberi (diploid D1) and G. mustelinum (tetraploid AD4) were studied for the identification of begomoviruses and associated satellites. We characterized begomoviruses, betasatellites and alphasatellites, performed comprehensive phylogenetic analysis, and identified a close relationship with the recent cotton-infecting begomovirus complex from Pakistan and India [begomovirus and satellites. The importance of our findings with reference to recent literature is also discussed.In this study, three non-cultivated cotton species nd India ,27. We aG. mustelinum, and diploid (D) G. raimondii and G. thurberi species  of RCA-enriched products detected diverse begomoviruses and associated satellites in tetraploid (AD)  species .G. mustelinum, G. raimondii and G. thurberi samples, respectively. The trimmed reads had a length distribution of ~151 in all libraries. The contigs of size ~2.8 kb and ~1.4 kb were likely to be begomovirus and alpha/betasatellites, respectively, because of their corresponding genome sizes. A mixed approach of de novo and reference-based assembly was used for the assembly of begomovirus/alphasatellite/betasatellite genomes. For reference-guided assembly, a comprehensive list of begomoviruses and satellites was prepared based on the information available from ICTV (www.ictvonline.org). Selected contigs from assemblies were checked for the presence of putative open reading frames (ORF) using NCBI ORF finder (www.ncbi.nlm.nih.gov/gorf). All contigs of ~2.8 kb length had ORF and genetic arrangement typical of DNA-A and/or DNA-B components of begomoviruses , cotton leaf curl Alabad virus (CLCuAlV) and bhendi yellow vein mosaic virus (BYVMV) in the non-cultivated cotton species under study . It must be mentioned here that in the literature, the name Gossypium punctatum mild leaf curl virus (GPMLCuV) has also been used for this virus, that later became CLCuAlV after the revised begomovirus taxonomic criteria [Jatropha curcas mosaic virus (JCMV), suggesting its close relationship with these and other related virus species. This observation is consistent with previous findings [We also detected the DNA-B component of bipartite begomoviruses, associated with CLCuAlV . All DNAcriteria . In thisfindings . G. mustelinum, and not in diploid G. thurberi and G raimondii. In G. mustelinum, five distinct species of alphasatellites were detected namely, Gossypium darwinii symptomless alphasatellite (GDarSLA), Gossypium davidsonii symptomless alphasatellite (GDavSLA), ageratum yellow vein Singapore alphasatellite (AYVSA), cotton leaf curl Burewala alphasatellite (CLCuBuA) and papaya leaf curl alphasatellite (PaLCuA)  and G. hirsutum (KX656847-KX656848) from Pakistan. Other alphasatellites species identified in this study showed high sequence identity (93%–100%) to PaLCuA, AYSVA and CLCuBuA sequences isolated from the Indian subcontinent. BLASTn results demonstrated that amino acid alignment of alphasatellite Rep protein described two variants; a Rep protein with 315 amino acids and another Rep like protein with 295 amino acids (S1 File). This kind of natural alphasatellite Rep variant has also been identified previously [Surprisingly, alphasatellites were only detected in tetraploid (PaLCuA) . SimilarG. thurberi. BLASTn analysis indicated the closest identity (95%–98%) with cotton leaf curl Multan betasatellite (CLCuMuB)-Shahdadpur strain  and -Burewala strain (accession no. KX697599) isolated from Vehari-Punjab-Pakistan [begomovirus-associated betasatellites  [G. lobatum, G. punctatum and G. gossypoides in 2012 [G. hirsutum during the recent outbreaks in India and Pakistan that confirmed the authenticity of our phylogenetic analysis. Taken together, CLCuMuV isolates harbored by non-cultivated cotton species since 2000s, are the possible progenitor CLCuMuV isolates now prevailing in recent outbreaks in cultivated cotton fields.A phylogenetic analysis was performed on the full-length sequences of DNA-A (and monopartite begomoviruses), DNA-B, alphasatellites and betasatellites with their respective similar isolates available in the database, based on MUSCLE alignment. A neighbor-joining (NJ) phylogenetic tree of DNA-A, reconstructed with DNA-A sequences in this study and other related ups A–C; a. Group akistan)   reported from Pakistan. Sequences in the clade II consisted of sequences isolated from G. mustelinum and G. raimondii, rooted with Thanagan strain of BYVMV (accession no. FJ176235) isolated from India. Detection of BYMV species from non-cultivated cotton species in this study is distinct from all the previous key reference studies [Group C in the phylogenetic tree of DNA-A consisted of BYVMV isolates that sub-grouped further into two clades a. Sequen studies ,27,31, sG. davidsonii in 2012, and therefore named as GDavSLA [G. darwinii (subclade I) and G. hirsutum (subclade II) from Pakistan [G. hirsutum from Pakistan and India, co-segregated indicating their close similarity . The other three alphasatellite isolates segregated with CLCuBuA, PaLCuA and AYVSA, previously reported from croton and cotton  and India (KY018415) [G. hirsutum from Pakistan  and India  [begomovirus (CLCuMuV) and betasatellite (CLCuMuB) may have occurred from non-cultivated to cultivated cotton species. The other clades in the phylogenetic tree represent other strains of CLCuMuB prevailing in the Indian subcontinent.The phylogenetic dendrogram of betasatellites segregated into two groups b; the fiY018415) ,27. The Y018415) , isolateY081414) ,27, whicbegomovirus, a quantitative analysis was performed using qPCR. Analysis indicated that the amount of begomovirus in G. raimondii and G. thurberi was one and a half times as much as that in the CLCuD infected cultivated cotton G. hirsutum; whereas in G. mustelinum, virus titer was five times less than that in G. hirsutum  and betasatellite (CLCuMuBBur) were identified as dominant causal agents [begomovirus tomato leaf curl New Delhi virus (ToLCNDV) is spreading widely in the cotton growing areas of Punjab and Sindh provinces in Pakistan [Cultivated cotton st CLCuD ,36. The st CLCuD . Howeverl agents . RecentlPakistan ,39. ThisPakistan  and IndiPakistan . The actPakistan , but thePakistan , suggestPakistan .We hypothesized that, since non-cultivated cotton species have been reported to maintain viruses from the first epidemic , they maG. raimondii which was not previously evaluated for the presence of begomoviruses. Begomoviruses or satellites were not detected in G. thurberi in the 2012 study [begomovirus species  were detected in G. raimondii. In G. thurberi, three begomovirus species with four distinct strains  were identified in association with two betasatellites species, CLCuMuB and OLCuB  is quite surprising. Since virus and satellite molecules from both recent studies also have phylogenetic relatedness among them, there must be a common region from where all these virus and satellite species are being harbored and spread to the nearby cotton fields whenever the environmental conditions are favorable. This study identified a common origin and signified the idea that non-cultivated cotton species may act as reservoirs for CLCuD-associated begomoviruses/satellites and could play a role in the reemergence of CLCuMuV, CLCuAlV and CLCuKoV in cotton fields of Pakistan and India in 2015. Apart from the strong phylogenetic relationship among begomoviruses and satellites reported here and recently from Punjab-Pakistan/India, they also have high sequence identity with the isolates previously reported from non-cultivated cotton species in 2012 by Nawaz-ul-Rehman et al. . This suG. mustelinum (AD4), G. raimondii (D5) and G. thurberi (D1), maintained at the Central Cotton Research Institute (CCRI) Multan , were collected in 2015 . Total DNA was extracted from 100 mg of leaf tissues using the CTAB method [For the molecular detection of possible virus/satellite, leaf samples from  in 2015 . Leaf saB method . RCA wasB method . RCA enrbegomovirus was made using a comprehensive list of begomoviruses prepared based on the information provided at the International Committee on Taxonomy of Viruses (ICTV) website (www.ictvonline.org). For reference-guided assembly of alphasatellite and betasatellite, sequences present in GenBank were used. All sequences were searched for similarity against the NCBI non-redundant nucleotides database (nt) using the BLAST + tool, provided by NCBI” [The details, from library preparation to the data analysis, have been described elsewhere . “The Ilby NCBI” .begomovirus DNA-A isolates, a neighbor joining phylogenetic tree was inferred from aligned sequences of begomoviruses  isolated in this study together with 235 other sequences from seven major cotton infecting monopartite begomovirus species with their representative strains reported so far in OW and New World (NW). For alphasatellites, neighbor joining dendrogram was constructed using 27 representative alphasatellite species reported from OW and NW along with alphasatellite sequences reported here. Neighbor joining phylogenetic tree for DNA-B isolates was inferred using aligned sequences isolated here together with other DNA-B sequences of 19 representative bipartite begomovirus species reported from Asia, Africa and Brazil. Similarly, betasatellite phylogenetic dendrogram was inferred using aligned betasatellite sequences isolated here along with the representative strains of CLCuMuB and OLCuB reported so far. For begomovirus species and strain demarcation, sequence demarcation tool (SDT) v1.2 was used to perform MUSCLE alignment and construct identity matrices following the methodology explained earlier [Sequence fasta files were imported in MEGA6 for alignment and phylogenetic analysis. All the sequences were aligned using MUSCLE option in MEGA6 . Same al earlier .p value 0.05.For the detection of any recombination events, recombination analysis was performed using virus sequences isolated in this study along with representative begomoviruses sequences used earlier  with theG. hirsutum DNA samples were used as positive and negative controls, respectively. We have provided a detailed qPCR methodology, adapted specifically for the detection of CLCuD complex in cotton, elsewhere [begomovirus genome or DNA-A component, DNA_B_qPCR_Forward (GCCCATGATTCGTTCGGAC)/DNA_B_qPCR_Reverse (CACGTGGTACTGGAATATCGCA) for DNA-B and Betasatellite_qPCR_Forward (GATTTGACTTATATTGGGCCAATTTAAT)/Betasatellite_qPCR_Reverse (GATACTATCCACAAAGTCACCATCGCTAAT) for betasatellites [Genomic DNA of non-cultivated cotton species was used in qPCR experiment, where CLCuD-infected and CLCuD-free lsewhere . “The prtellites . qPCR retellites .


Vibrio parahaemolyticus can cause gastrointestinal illness through consumption of seafood. Despite frequent food-borne outbreaks of V. parahaemolyticus, only 19 strains have subjected to complete whole-genome analysis. In this study, a novel strain of V. parahaemolyticus, designated FORC_022 (Food-borne pathogen Omics Research Center_022), was isolated from soy sauce marinated crabs, and its genome and transcriptome were analyzed to elucidate the pathogenic mechanisms. FORC_022 did not include major virulence factors of thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). However, FORC_022 showed high cytotoxicity and had several V. parahaemolyticus islands (VPaIs) and other virulence factors, such as various secretion systems , in comparative genome analysis with CDC_K4557 (the most similar strain) and RIMD2210633 (genome island marker strain). FORC_022 harbored additional virulence genes, including accessory cholera enterotoxin, zona occludens toxin, and tight adhesion (tad) locus, compared with CDC_K4557. In addition, O3 serotype specific gene and the marker gene of pandemic O3:K6 serotype (toxRS) were detected in FORC_022. The expressions levels of genes involved in adherence and carbohydrate transporter were high, whereas those of genes involved in motility, arginine biosynthesis, and proline metabolism were low after exposure to crabs. Moreover, the virulence factors of the type III secretion system, tad locus, and thermolabile hemolysin were overexpressed. Therefore, the risk of foodborne-illness may be high following consumption of FORC_022 contaminated crab. These results provided molecular information regarding the survival and pathogenesis of V. parahaemolyticus FORC_022 strain in contaminated crab and may have applications in food safety. Vibrio parahaemolyticus is curved rod-shape, Gram-negative halophilic bacterium with a single polar flagellum and is abundant in estuarine environments and various seafood, including shellfish, oysters, clams, cockles, crabs, and shrimps  database1. shrimps . This ba shrimps . Recentlhe world ,b. The she world . To undeoisoning . The comVibrio parahaemolyticus strains isolated from patients hospitalized due to food-borne illness harbor multiple virulence genes, including thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh) , which harbored tdh gene as major virulence factor, caused food poisoning in Japan , which are located in pathogenic islands in V. parahaemolyticus, are thought to play a role in the pathogenicity of this organism  . These gal cells . For exain Japan . However factors . Genes rgenicity . Variousmembrane . In addiorganism . Infectitoxicity . TherefoV. parahaemolyticus attributed to a single food commodity are due to aquatic products, such as crustaceans and shellfish  NaCl for 12 h .The cells were negatively stained with uranyl acetate for 1 min and were then observed using a transmission electron microscopy  at 200 kV. The FORC_022 strain was a curved, rod-shaped bacterium that was 1.5–2.0 μm in length and 0.6–0.8 μm in width with a single polar flagellum  were infected with FORC_022 or KCTC 2471 (control) at various multiplicities of infection (MOI) for 4 h. The MOI is the ratio for the number of bacterial cells to the number of epithelial cells. V. parahaemolyticus KCTC 2471 strain, which was isolated from patients of food poisoning in Japan, was used as control in LDH assay  released from INT-407 human epithelial cells  after the plasma membrane was damaged. INT-407 cells were grown in minimum essential medium containing 1% (v/v) fetal bovine serum  in 96-well culture dishes  as described previously . Cells  according to the manufacturer’s protocol. Contamination of pure cultured strain was verified using 16S rRNA sequencing, and taxonomic identification was conducted by phylogenetic tree analysis using MEGA6 . The phy2 (Open reading frames (ORFs) and annotations were predicted by the Rapid Annotation using Subsystem Technology (RAST) server  and Gene2 .V. parahaemolyticus strains  based on ANI values  in V. fischeri minimal medium containing glycerol and then exposed to crab for 4 h. The V. fischeri minimal medium containing glycerol was used for artificial seawater medium for Vibrio . Total RNAs were isolated using a miRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. DNA contaminations were removed using TURBO DNase , and extracted RNAs were then cleaned up using an RNeasy MinElute Cleanup kit (Qiagen). The extracted RNA was verified with an Agilent 2100 Bioanalyzer with Agilent RNA 6000 Nano reagents . The cDNA library was constructed from enriched mRNA using a TruSeq Stranded mRNA Sample Preparation kit (Illumina) following the manufacturer’s instructions. The quality of cDNA libraries was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies). Strand-specific paired-ended 100-nucleotide reads from each cDNA library were obtained using HiSeq2500 (Illumina). For biological replication, two libraries were separately constructed and sequenced from RNAs isolated from two independently filtered culture supernatants of FORC_022.To sequence the RNA, mRNA was enriched from extracted total RNA by depleting rRNAs by using a Ribo-Zerop-values < 0.01 were considered differentially expressed in samples using the CLRNASeqTM program (ChunLab). Transcriptome analysis and visualization of virulence gene data were carried out using Gitools (Reads obtained from RNA sequencing were mapped to the FORC_022 reference genome using CLC Genomics Workbench ver. 7.5.1 (CLC Bio). The relative transcript abundance was measured by reads per kilobase of transcript per million mapped sequence reads . Genes w Gitools .recA expression level as an internal reference for normalization (t-tests in SigmaPlot software (ver. 12.0).Extracted RNA was converted to cDNA for qRT-PCR using an iScript cDNA Synthesis Kit . Real-time PCR of cDNA was performed using a Chromo 4 Real-time PCR Detection system (Bio-Rad) with SYBR Green I . The prilization . The amplization . qRT-PCRV. parahaemolyticus FORC_022 was deposited in the GenBank of NCBI4 under accession numbers CP013248, CP013249 and CP013250 for chromosomes I, II, and a plasmid, respectively. Transcriptome analysis data of virulence factors in V. parahaemolyticus FORC_022 was deposited in the NCBI Sequence Read Archive (SRA) under the accession number SRP135619.The whole genome sequence of V. parahaemolyticus by phylogenetic tree analysis of the 16S rRNA gene . However, FORC_022 did not encode major virulence factors such as tdh and trh genes, which are correlates with the Kanagawa phenomenon and induce significant cytotoxicity  strain as a positive control, which was isolated from a food poisoning patient . The cyttoxicity . Most stutbreaks . The TDHtoxicity . Howeverrh genes . In addigenicity . ConsistDatabase  and annoDatabase .V. cholerae) (V. cholerae) (V. parahaemolyticus in eukaryotic cells (V. parahaemolyticus has two icmF family genes (icmF1 and icmF2) in type VI secretion systems, which contribute to pathogenicity including adhesion to host epithelial cells and cytotoxicity  were detected; these genes may be responsible for the virulence of the FORC_022 strain. In addition, various secretion systems including types I, II, III, IV, and VI  were detected in the genome sequence of the FORC_022 strain. Type I secretion systems were detected from FORC22_0395, FORC22_1432, FORC22_1615-1620, and FORC22_2067 in chromosome I and FORC22_4335, and FORC22_4733-4736 in chromosome II; these secretion systems are required for the secretion of repeat-in-toxin  . Type IIholerae) . Type IIholerae) , were deic cells . Moreoveic cells , were deic cells ; these rtoxicity . The icmV. parahaemolyticus leading to its pathogenicity. Previous study reported that iron loss caused by V. parahaemolyticus affects the integrity of heme protein and leads to host cell death  was obtained based on ANI values . The highest ANI values (98.54%) were detected for FORC_022 and CDC_K4557 strain (Table 1), the latter of which was isolated from the stool of a patient in Louisiana in 2007 and was submitted to the Centers for Disease Control and Prevention (CDC)  . Signifimporters , whereasmporters . Another ruckeri . AlthougTable 2) . Three virulence factors of virulence-associated E, Type II/IV secretion systems, and Type IV pilin PilA were detected only in FORC_022. Therefore, VPaIs and additional virulence factors in FORC_022 may play important roles in the pathogenicity of this strain.VPaIs in FORC_022 and CDC_K4557 were identified by BLAST search against RIMD 2210633 strain, a clinical isolate used to identify genomic islands that could be markers for pandemic clones (Table 2) . Four VPter 1995 . VPaI-3 p-value < 0.01, two fold threshold; Supplementary Figure 5 with the FORC_022 genome as the reference database . The top 15 overexpressed genes (over 5-fold change) from FORC_022 exposed to crabs are summarized in Table 3, and the top 15 down-regulated genes are presented in Supplementary Table To compare gene expression in strains with or without contact with crabs, FORC_022 was exposed to washed swimming crabs in inoculation with minimal medium  and incubated for 4 h. The crabs tended to decompose after 6 h; thus, we analyzed samples after 4 h of incubation. Numerous genes were differentially expressed with significance in volcano plot (R2) ranging from 0.919 to 0.999. In addition, virulence factors, such as the type III secretion system, which exhibited cytotoxic activity toward human cells and was related to inflammatory diarrhea and septicemia (Table 3).The expression levels of genes related to amino acid transport and metabolism and lipid transport and metabolism were lower in the contact strain than in the strain not exposed to crabs, whereas the expression levels of genes related to carbohydrate transport and metabolism were higher in strains after contact with crabs. Genes related to biofilm formation such as tad locus, capsular polysaccharide (CPS), and lipopolysaccharide (LPS) , were ovpticemia , were alp-value < 0.01; Figure 5A). This result showed that genes related to virulence factors, such as the EPS type II secretion system, type III secretion system, MSHA type IV pilus, thermolabile hemolysin (tlh), and heme receptors, were overexpressed in the contacted strain . The levels detected by real-time PCR were consistent with the transcriptome results. These results suggested that the potential risk of foodborne illness by ingestion of contaminated crab with FORC_022 could be high due to its virulence factors.A comparison of expressed virulence genes between strains with and without contacted with crabs is presented in the form of a heatmap , suggesting that other virulence factors may play a role in the pathogenesis of this infection. FORC_022 had additional virulence factors, such as accessory cholera enterotoxin, zona occludens toxin, and tad locus, compared with CDC_K4557 (the strain most closely related to FORC_022). In addition, the pandemic O3:K6 serotype specific gene (toxRS) and VPaI-1 were detected in FORC_022. Expression levels of adherence-, carbohydrate transporter-, and biofilm formation-related genes increased simultaneously after FORC_022 was exposed to crabs (Table 3). Therefore, this strain could survive and grow on crabs. In addition, several virulence factors, including the type III secretion system, tad locus, and thermolabile hemolysin, were overexpressed ; this could lead to a higher risk of infection and pathogenicity (V. parahaemolyticus in the crab products. Further studies including immunology analysis and animal model experiments are necessary to verify the pathogenicity of FORC_022 in present study.In the present study, we analyzed the complete genome sequences of genicity . AlthougK-HL, SR, HC, J-HL, HBK, HK, HJ, SC, and B-SK designed the study. HC, BL, and B-SK analyzed the sequencing data. HC, BL, and EN performed the experiments. HC, K-HL, SR, HY, J-HL, HBK, HK, HJ, SC and B-SK discussed the results and wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vibrio cholerae is the causative agent of cholera, a life-threatening diarrheal disease. Cholera causes epidemics and pandemics, but the ways this disease spreads worldwide is still unclear. This review highlights a relatively new hypothesis regarding the way V. cholerae can be globally dispersed. Copepods and chironomids are natural reservoirs of V. cholerae and are part of different fish species’ diet. Furthermore, V. cholerae inhabits marine and freshwater fish species. Waterbird species feed on fish or on small invertebrates such as copepods and chironomids. Waterbirds have also been found to carry living copepods and/or chironomids internally or externally from one waterbody to another. All of the above points to the fact that some waterbird species might be vectors of V. cholerae. Indeed, we and others have found evidence for the presence of V. cholerae non-O1 as well as O1 in waterbird cloacal swabs, feces, and intestine samples. Moreover, hand-reared cormorants that were fed on tilapia, a fish that naturally carries V. cholerae, became infected with this bacterial species, demonstrating that V. cholerae can be transferred to cormorants from their fish prey. Great cormorants as well as other waterbird species can cover distances of up to 1,000 km/day and thus may potentially transfer V. cholerae in a short time across and between continents. We hope this review will inspire further studies regarding the understanding of the waterbirds' role in the global dissemination of V. cholerae. Vibrio cholerae, and thus may distribute this species all over the globe.Birds are ubiquitous and globally distributed. There are 10,000 known bird species, which account for over 15% of all vertebrates ,3. Only V. cholerae is a gram-negative, facultative anaerobe, motile curved rod. It belongs to the family of Vibrionaceae and is the etiological agent of cholera, a life-threatening disease. Strains belonging to V. cholerae inhabit both marine and freshwater ecosystems [V. cholerae serogroups, but only serogroups O1 and O139 have been associated with cholera endemics and pandemics [V. cholerae non-O1/O139 strains can also cause intestinal and extra-intestinal infections such as gastroenteritis, cholera-like diarrhea, wound infections, external otitis, and bacteremia that sometimes can be fatal in humans [osystems . There aandemics . V. choln humans –18.V. cholerae serogroups O1 or O139 by consuming contaminated food or water. Toxigenic strains cross the human gastric acid barrier and then colonize the small intestine epithelial cells. After colonization, the bacterium produces the cholera toxin, which triggers fluid secretion by the intestinal epithelium, causing acute dehydration [Cholera is a severe diarrheal disease that has afflicted human beings and shaped human history for over 2 millennia ,18. The ydration ,19. WHO ydration .V. cholerae strains from different geographic regions suggested that the bacterium was introduced into Haiti from Nepal by humans [V. cholerae strains from the Yemen epidemic with strains from Asia and Africa. They concluded that the source of the epidemic strains in Yemen is a strain related to a cholera outbreak in South Asia first detected in 2012. However, this strain, which entered Yemen in 2016, had been circulating and causing outbreaks in eastern Africa in 2013 through 2014 before it appeared in Yemen in 2016 [An example of a cholera epidemic is the outbreak in Haiti in October 2010. Diverse studies pointed out that the clinical isolates from that event were most closely related to Asian isolates . Studiesy humans –24. Sincy humans . Weill ay humans  compared in 2016 .V. cholerae is part of the normal microbial population and ecology of the surface water of our planet. Colwell and colleages [V. cholerae proliferates while attached to or associated with eukaryotic organisms in the aquatic environment, particularly copepods (Crustacea). We found indications that chironomids serve as reservoirs for V. cholerae [Diptera) are one of the most widely distributed insects in marine and freshwater habitats. V. cholerae was isolated from all 4 life stages of chironomids [V. cholerae can survive better in seawater when it is associated to zooplankton than as a free cell [olleages –30 showecholerae –38. Chirronomids . It has ree cell .Acanthamoeba castellanii [V. cholerae.In laboratory studies, serogroups O1 and O139 were able to grow and survive in the cytoplasm of trophozoites and in the cysts of free-living amoeba tellanii . Arthroptellanii , oysterstellanii , cyanobatellanii  were alsV. cholerae in fish intestines in various water habitats in Israel. They found 11 fish species that inhabited V. cholerae (10 species from fresh water habitats and one from a marine habitat). One species (tilapia) harbored 5 × 103 colony-forming units (cfu) per 1 g intestinal content. They suggested that fish can be a reservoir of V. cholerae and can act as small-scale vectors for the dispersal of this bacterium [V. cholerae in the fish gut. Correlation of some cholera outbreaks with the consumption of uncooked fish has been reported [V. cholerae may have a role in chitin degradation. Thus, the fish host and V. cholerae may have a commensal relationships [Senderovich and colleagues  surveyedacterium . Fish coreported –48. Sendreported  suggesteionships .V. cholerae. V. cholerae was isolated from fish intestines, gills, skin, kidney, liver, and brain tissues, and in total, were identified in 30 fish species [V. cholerae O1 and showed that the bacteria could attach to a fish’s intestinal epithelium and form microcolonies. They also showed that contaminated fish could spread the bacterium to naive fish.Halpern and Izhaki  reviewed species . In most species  infectedV. cholerae in fish has accumulated. For example, Hossain and colleagues [Tenualosa ilisha) to act as a vector of V. cholerae to humans. This fish migrates from cholera-endemic areas to freshwater rivers around Bangladesh and is the most consumed fish species in that country. They found that about 16% of their isolates (n = 158) were V. cholerae O1 strains [V. cholerae O1 was also isolated from the gills of a freshwater fish [V. cholerae non-O1/O139 isolates were identified from Malaysian fish [Recently, more evidence on the presence of lleagues  studied  strains . V. cholter fish  and fromter fish . Fifty-tian fish  and fromian fish .V. cholerae to cross water bodies and even oceans. Huq and Colwell [V. cholerae cells are dispersed in the water while attached to copepods and this serves as a mechanism for its global distribution. However, humans consume freshwater whereas the copepods’ journey between continents occurs in the ocean (marine water). Broza and colleagues [Cholera spreads all over the globe and causes epidemics and pandemics. Nevertheless, despite intensive research efforts, its ecology remains an enigma, in particular the mechanism that enables  Colwell  suggestelleagues  suggesteV. cholerae within and between continents. They suggested that the bacterium can pass from endemic to uninfected water bodies via waterbirds in 2 courses: (i) waterbirds may carry directly contaminated copepods and/or chironomids  –59. Therronomids ; (ii) waronomids . Their hspersion .V. cholerae in waterbirds. These studies had become overlooked and forgotten over the years. Bisgaard and Kristensen [V. cholerae from 2 ducklings at a Danish duck farm , great black-backed gulls (Larus marinus), herring gulls (Larus argentatus), and rooks (Corvus frugilegus). In 1980, V. cholerae was isolated from the liver and heart of a domestic goose (Anser anser) [V. cholerae was isolated from cloacal swabs and fresh droppings that were collected from 20 waterbird species [V. cholerae non-O1/O139 were detected in coots, cormorants, gadwalls, gulls, herons, killdeers, mallards, pelicans, pintails, teals, redheads, egrets, ibises, and phalaropes species [n = approximately 200) were classified into 22 different serogroups  [V. cholerae serogroup O1 biotype El Tor Ogawa was isolated from cloacal swabs and fresh feces of the great blue heron (Ardea herodias) and ring-billed gull (Larus delawarensis). Unstable O1 serogroup was detected from 3 other waterbird species: California gull , American coot (Fulica Americana), and a double-crested cormorant . This means that out of the 20 studied waterbird species, 5 (25%) carried pathogenic serogroups [V. cholerae non-O1 was also identified from gulls that were sampled in Connecticut [Halpern and colleagues  also fouistensen  isolateduck farm . Lee andr anser)  (Table 1 species . V. chol species  (Table 1rogroup)  (Table 1rogroups  (Table 1necticut .V. cholerae isolation from different waterbird species strongly support the hypothesis that migratory waterbirds may serve as vectors for V. cholerae [All of these findings from relatively old published literature (before 1989) regarding cholerae ,60.V. cholerae [V. cholerae in waterbirds have been reported. In Venezuela, V. cholerae O1 Inaba El Tor and V. cholerae non-O1 were isolated and identified from 6 greater yellowlegs (Tringa melanoleuca) and from 6 Wilson's plover (Charadrius wilsonia), respectively [Vibrio spp. in fecal samples of resident and migratory waterbirds around 2 costal sites in the tropical southern Caribbean Sea, Venezuela. They isolated V. cholerae from 5 waterbird species: sandpipers , large-billed tern (Phaetusa simplex), American flamingo (Phoenicopterus ruber), wattled jacana (Jacana jacana), and semipalmated sandpiper  [V. cholerae non-O1/non-O139 was isolated from cloacal swab samples of wild manx shearwater (Puffinus puffinus) that were caught in the north-central coast of Rio de Janeiro, Brazil [V. cholerae non-O1/non-O139 isolates from waterbird species: kelp gull (Larus dominicanus), Atlantic yellow-nosed albatross , brown booby (Sula leucogaster), Cabot’s tern , and grey-headed gull     .V. cholerae non-O1/O139 from the intestines of 1 out of 7 wild great cormorant  individuals sampled in Israel. They detected by molecular tools the presence of ompW gene in 5 individual cormorants, demonstrating the presence of V. cholerae in 5 out of 7 wild cormorants. The presence of cholera toxin subunit A (ctxA) and serogroup O1 was also molecularly detected in the intestine of 3 and 1 individual cormorants, respectively (V. cholerae was detected in the intestine of 3 wild waterbird species in Israel: little egret (Egretta garzetta), black-crowned night heron (Nycticorax nycticorax), and black-headed gull (Larus ridibundus). Forty-six V. cholerae isolates were obtained from the intestines of little egrets and black-crowned night herons. These isolates were classified into 23 different serogroups . All isolates were found positive for toxR gene and negative for ctxA, tcpA, tcpI, zot, and ace genes. In addition, hapA was found in 95.3% of the isolates, hlyA in 93.0%, ompU in 41.0%, and 9.7% were found positive for some of the type three secretion system (TTSS) genes  [V. cholerae serogroups inhabit the intestine of an individual. Although V. cholerae was not isolated from black-headed gulls, the presence of ompW gene, which identifies the presence of V. cholerae, was detected in 1 out of 5 black-headed gull intestine samples, indicating that V. cholerae was present in this bird species  . More th species . Fig 2.  species . Fig 2.V. cholerae by consuming naturally colonized fish. They tested 8 captive hand-reared great cormorants, divided into 2 groups: (i) the control group, which fed on golden fish that was negative to the presence of V. cholerae and (ii) the experimental group that fed on tilapia that is naturally colonized by V. cholerae. In the control group, both the fish and the cormorants were negative to V. cholerae throughout the 3 weeks of the experiment. In the experimental group, V. cholerae was transferred from the naturally colonized tilapia to the cormorants and was detected in the cormorants’ feces. They also demonstrated that V. cholerae could survive in the cormorants’ digestive tract even 72 hours after tilapia ingestion. According to the literature, in a period of 72 hours, great cormorants are able to cross oceans [Laviad-Shitrit and colleagues  studied V. cholerae strains isolated from waterbird intestine samples. They found that waterbirds showed the highest minimal inhibitory concentration (MIC) values to all studied antimicrobial agents (except ampicillin) compared with strains isolated from fish or chironomids [V. cholerae resistant to ampicillin from the brown booby (Sula leucogaster). Hence, waterbirds may also be vectors for antimicrobial resistant strains and may spread them globally. It is the responsibility of local administrations to monitor areas with large migratory waterbird populations for V. cholerae presence and for antimicrobial resistance properties of the bacteria.Laviad-Shitrit and colleagues  studied ronomids . Cardosoronomids  isolatedV. cholerae infection  producing V. cholerae, should serve to answer this question. This should also be followed by observing the birds’ intestines to find out whether V. cholerae is attached to the intestines’ epithelial cells. If the bacteria colonize the intestine, are they transferred horizontally or vertically to their offspring? Does cholera toxin have some functions in waterbirds? Can we determine a model waterbird species that carries V. cholerae? Can we use this model to study and understand the role of V. cholerae pathogenic genes? Can we use this waterbird species model to monitor the dissemination of epidemic V. cholerae strains from one location to another and perhaps eventually to predict and even take measures to prevent cholera outbreaks?Does S1 TableThe list specifies the phylogenetic position of each bird species. All waterbird species belong to the class Aves in the phylum Chordata.(DOCX)Click here for additional data file.


The greater the diversity, the greater the perfection.”“Thomas Berry—A centromere is classically defined as the primary constriction on a metaphase chromosome  that holCandida, which harbors several pathogenic species, presents a diverse array of centromere types. Candida glabrata carries point genetic centromeres, much like the 125-bp DNA sequence that serves as a fully functional point centromere in the budding yeast Saccharomyces cerevisiae [C. glabrata are not fully functional in S. cerevisiae, suggesting that centromere function is species-specific [Candida tropicalis comprise a central core flanked by inverted repeats, similar to those of the fission yeast Schizosaccharomyces pombe [C. tropicalis can acquire a hairpin loop-like secondary structure that might be crucial for kinetochore assembly. Candida albicans and Candida dubliniensis, on the other hand, possess unique and different centromere DNA sequences on each of their chromosomes [C. tropicalis centromeres can stabilize an ARS plasmid, indicating a role of DNA sequence in centromere identity, the same does not hold true for C. albicans. Cryptococcus neoformans, a basidiomycetous pathogen that diverged from C. albicans more than 900 million years ago, harbors large regional centromeres that are rich in centromere-specific retroelements [Candida lusitaniae (teleomorph Clavispora lusitaniae) and Malassezia sympodialis centromeres are present in highly AT-rich regions of the genome but lack any easily detectable conserved sequence motifs or repeats [DNA sequence features provide the necessary template to act as a binding platform for kinetochore proteins. The genus revisiae –5. Typicspecific . Short res pombe , 7. The omosomes , 9. Whilelements –12. Whil repeats , 14. Whe repeats , remainsMucor circinelloides and Phycomyces blakesleeanus are notable exceptions in this regard, as they have no obvious CENP-A homologs, even though their centromeres are not yet physically mapped [C. albicans. Like other species, the CENP-A chaperone Holliday junction recognition protein/suppressor of chromosome mis-segregation 3 (HJURP/Scm3) is found to be crucial for CENP-A loading in C. albicans (our unpublished results). Regional centromeres harbor canonical histone H3 along with CENP-A. Post-translational modifications of histone H3 are crucial in forming a functional kinetochore [C. neoformans [C. neoformans, but its functional significance is unclear yet [The conserved centromere-specific histone H3 variant CENP-A/Cse4 is specifically present at all fungal centromeres identified to date but is largely excluded from other regions of the genome. y mapped . CENP-A y mapped . Althougy mapped . The proetochore . Biochemoformans . Apart flear yet .C. glabrata and Ustilago maydis, have lost all of the proteins required for functional RNAi during the course of evolution, whereas species including C. albicans and C. tropicalis harbor a cryptic RNAi machinery [Cryptococcus species complex correlated the loss of RNAi with the length of centromeres, thereby proposing that RNAi helps in maintaining long repetitive, transposon-rich centromeres [Cryptococcus and Ustilago species complex [For a very long time, the centromere locus was considered heterochromatic and transcriptionally inert. While centromere regions are generally transcription poor, landmark studies in several yeast species revealed that small interfering RNAs (siRNAs) derived from pericentromeric regions are necessary for centromere function , 21. Theachinery . A recentromeres . Whethertromeres , 24. How complex .C. albicans, centromeres replicate early in every synthesis phase (S-phase) and are associated with an early firing replicating origin [C. albicans and load it at the site of stalled replication forks; that is, centromeres. How this process is regulated to occur only during S-phase remains unknown, with possible implications for the CENP-A chaperone Scm3. Based on studies in many other nonpathogenic fungal species, the physical proximity of a partitioning locus (centromere) and an initiator site (replication origin) is relevant when one dissects the functional aspects of genome maintenance. However, evidence toward this connection is just beginning to emerge.Unlike metazoans, most fungal species have early replicating centromeres –27. Thisg origin . Additiog origin . CentromC. albicans, C. tropicalis, and Fusarium graminearum. Using chromosome conformation capture (3C) experiments, clustered centromere DNA regions were shown to be present in close spatial proximity, leading to physical interactions between different centromeres [S. cerevisiae, for example, a locally enriched population of accessory CENP-A molecules at pericentric chromatin has been shown to serve as a reservoir for rapid incorporation of CENP-A in the event of premature eviction from centromeres [C. albicans in which neocentromeres were formed close to the native centromere in most cases [C. albicans change the spatial location to be a part of the centromere cluster by 3C experiments [C. neoformans cells that eventually cluster at the onset of mitosis [Most fungal centromeres are clustered near spindle pole bodies (SPBs). This association may result in folding back of chromosomes and positioning them such that telomeres are juxtaposed in the interphase nucleus, giving rise to the Rabl conformation . This phtromeres –32. It htromeres , 34. It tromeres . Furthereriments . Interes mitosis . WhetherC. neoformans, CENP-A assembly initiates the loading of other proteins, giving rise to a sequential kinetochore assembly in this organism [C. albicans shows an interdependent kinetochore formation in which probably all kinetochore proteins assemble as a single complex [Candida and Cryptococcus species complexes revealed that centromere-mediated recombination might have contributed to variations in the centromere structure and sequence [C. albicans and C. dubliniensis centromere sequences show a high level of divergence while the rest of the genome sequences are highly homologous [Candida and Cryptococcus species as well as the application of efficient and improved DNA sequencing technologies for obtaining better genome assemblies are some of the significant recent developments that will advance pathogenic fungal molecular genetic research.Overall, here we summarize five key determinants among many that are needed for centromere identity in fungal pathogens . These forganism . On the  complex . Howeversequence , 10, 38.mologous . Discove


Propionibacterium acnes is a rare etiology of HP, and its pathology remains unclear.Hypertrophic pachymeningitis (HP) is a rare disorder that involves localized or diffuse thickening of the dura mater. HP is associated with various inflammatory, infectious, and malignant diseases, such as rheumatic arthritis, sarcoidosis, anti-neutrophil cytoplasmic antibody-associated vasculitis, IgG4-related disorders, syphilis, tuberculosis, bacterial and fungal infections, cancer, and idiopathic diseases, when evaluation fails to reveal a cause. Among them, chronic infection with An 80-year-old man having refractory otitis media with effusion of the right ear presented with progressive right-sided headache and nausea. Post-contrast brain magnetic resonance imaging revealed right mastoiditis and remarkable thickening of the dura mater and enhancement of pia mater extending from the right middle cranial fossa to the temporal lobe.P. acnes, and histopathological examination using P. acnes-specific monoclonal antibody (PAB antibody) revealed the infiltration of inflammatory cells with P. acnes. Moreover, using PAB antibody, P. acnes was detected in the biopsy specimen of the thickening dura mater. No granulomas were identified in either specimen. HP was resolved with long-term administration of antibiotics and steroids.HP secondary to middle ear infection was suspected, and a biopsy of the right mastoid was performed. An anaerobic culture of the biopsied right mastoid showed the growth of P. acnes infection followed by refractory otitis media. This report showed that chronic latent P. acnes infection induces chronic inflammation.This is the first documentation of pathologically demonstrated chronic HP associated with  Propionibacterium acnes (P. acnes) is a rare etiology of HP [Hypertrophic pachymeningitis (HP) is a rare disorder that involves localized or diffuse thickening of the dura mater . Most pagy of HP .P. acnes is a slow-growing, non-spore forming anaerobic gram-positive bacillus that ubiquitously resides in human skin and hair follicles [P. acnes can invade and persist in epithelial cells and circulating macrophages, thus inducing chronic inflammation [ollicles . Intriguammation ; it is cammation .P. acnes with the intracranial infections, it is one of the important pathogens responsible for postoperative meningitis and subdural empyema [P. acnes have been reported which were not associated with neurosurgical procedures or trauma [Regarding the association of  empyema –9. Howevr trauma –12. Morer trauma , 14.P. acnes infection, which was resolved by long-term administration of both antibiotics and steroids.Here we report the first case of HP due to pathologically proven chronic An 80-year-old man presented with deafness and otorrhea of right ear and was diagnosed as having otitis media with effusion (OME). Myringotomy, the insertion of a ventilation tube, and administration of multiple oral antibiotics (cefditoren pivoxil (CDTR-PI) 300 mg/day for 2 weeks, sitafloxacin (STFX) 100 mg/day for 19 days, and clarithromycin (CAM) 400 mg/day for 2 weeks) were only partially effective; his OME had been recurrent and refractory to these treatments. Although myeloperoxidase and proteinase 3 anti-neutrophil cytoplasmic antibodies  were negative in the blood examination, otitis media with ANCA-associated vasculitis was suspected, and prednisolone (30 mg/day) was orally administered  without Six months later, a chronic right-sided headache emerged and gradually worsened. Eight months after the onset of OME, he suffered from nausea and severe headache and was admitted to our hospital.3, mononuclear cells 30/mm3) and total protein levels (91 mg/dl). Repeated CSF cultures were negative, and the results of the polymerase chain reaction analyses performed on the CSF for Mycobacterium tuberculosis, Epstein–Barr virus, Cytomegalovirus, and Herpes simplex virus were also negative. CSF cytology was normal, and no oligoclonal band was detected. Neither cryptococcal antigen testing nor Indian ink staining of CSF was positive.Post-contrast MRI revealed right mastoiditis, remarkable thickening of the dura mater, and enhanced pia mater extending from the right middle cranial fossa to the temporal lobe Fig. . CerebroMycobacterium tuberculosis was not present as per the results of the interferon-gamma release assays. βD-glucan was not detected in the blood. C reactive protein levels were slightly elevated (1.19 mg/dl).Both MPO-ANCA and PR3-ANCA were evaluated again in the serum; however, the results were negative. An additional blood examination showed normal angiotensin-converting enzyme, lysozyme, and immunoglobulin G4 levels. Anti-CCP antibodies, rheumatoid factor, and anti-nuclear antibodies were not detected. Serology test results for syphilis, human immunodeficiency virus, human T-lymphotropic virus type 1, candida, and aspergillus were all negative. 3) and protein levels (58 mg/dl) on day 13, the patient’s headache gradually worsened, and a post-contrast MRI showed no improvement ; culture results for P. acnes were also positive  (6 g/day) and vancomycin (VCM) 2 g/day) were administered. Although the CSF examination showed decreased cell counts (19/mm g/day weP. acnes is sensitive to ceftriaxone (CTRX), MEPM and VCM were switched to CTRX (4 g/day). Although the patient received predonisolone (PSL), trimethoprim-sulfamethoxazole (TMP-SMX) was also prescribed for prophylaxis against pneumocystis pneumonia [To control the post-infectious intracranial inflammation, high-dose intravenous methylprednisolone (IVMP) (1000 mg/day) was administered for 5 days followed by oral prednisolone (45 mg/day), and his headache was remarkably alleviated. Because neumonia . PSL wasP. acnes was detected using PAB antibody; however, it was not detected in the culture of the dura mater  hematogenous routes via the diploic veins from the hair follicles or sebaceous glands of the scalp or (2) invasion from adjacent inflammation such as sinusitis, orbititis, and otitis media [P. acnes, are important pathogens in otitis media [P. acnes colonization and biofilm production; consequently, P. acnes subsequently invaded the adjacent intracranial meninges.CNS infections caused by lacement –9. Brainridement –9. By copromised  and immupromised –14. In Pis media  and are is media . In thisP. acnes has often been considered a common contaminant of blood and fluid cultures, we analyzed the biopsied mastoid by immunohistochemistry with PAB antibody to exclude the possibility that the P. acnes in the fluid cultures from the biopsied right mastoid was a consequence of false-positive contamination. In addition, although the CSF culture did not test positive for P. acnes, it was successfully detected by immunohistochemistry with PAB antibody within the biopsied dura mater and mastoid.Because P. acnes-associated neurosarcoidosis identified by immunohistochemical analysis of the brain tissue using PAB antibody [P. acnes antigens in the infected tissues putatively induced the cytotoxic immune response and exaggerated the inflammatory reaction, which cannot be controlled without the administration of steroids.Yang et al. reported a case of antibody . Contrarantibody . The mecP. acnes-associated CNS infection can be treated with antibiotics in most cases, French et al. reported a rare case of P. acnes-associated meningitis responsive to steroids [P. acnes [P. acnes in CSF during the treatment of chronic meningitis and it was unclear whether P. acnes actually caused the chronic meningitis, we should be aware that steroid administration against chronic P. acnes infection without antibiotics might result in a fatal outcome. Except for sarcoidosis, which has been reported to be caused by a hypersensitive Th1 immune response to P. acnes and can thus be treated by steroids, chronic P. acnes infection should be carefully observed when steroids are administered.Although steroids . ConversP. acnes . AlthougP. acnes-associated inflammation without either a histopathological examination or blood testing of ANCA in this patient.Notably, otitis media sometimes accompanies HP in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis without systemic manifestations such as pulmonary and renal involvement , 26. It P. acnes-associated chronic HP, future study which uncover the clinical course of multiple patients with the same disease will be needed.We should be aware that this report has limitations of being based on a single case: in order to determine the effectiveness of the treatment for P. acnes-associated chronic HP followed by refractory otitis media. The patient was successfully treated by long-term administration of antibiotics and steroids. Further study is necessary to reveal how P. acnes induce chronic latent infection and inflammation in the brain and establish treatment strategies to control the chronic inflammation.In conclusion, we report the first known case of


Amaranthus palmeri S. Watson), are problematic in row-crop producing areas of the United States. The objectives of this study were to determine if chlorimuron-ethyl, fomesafen, and glyphosate applied separately and in mixtures control A. palmeri and confirm the presence of various genotypes surviving two- and three-way herbicide mixtures. Fifteen percent of A. palmeri treated with the three-way herbicide mixture survived. Mixing fomesafen with chlorimuron-ethyl or fomesafen with glyphosate to create a two-way mixture reduced A. palmeri survival 22 to 24% and 60 to 62% more than glyphosate and chlorimuron-ethyl alone, respectively. Previously characterized mutations associated with A. palmeri survival to chlorimuron-ethyl, fomesafen, and glyphosate Trp574Leu, a missing glycine codon at position 210 of the PPX2L gene (ΔG210), and 5-enolpyruvylshikimate-3-phosphase synthase (EPSPS) gene amplification; respectively, were present in surviving plants. However, 37% of plants treated with chlorimuron-ethyl did not contain heterozygous or homozygous alleles for the Trp574Leu mutation, suggesting alternative genotypes contributed to plant survival. All surviving A. palmeri treated with fomesafen or glyphosate possessed genotypes previously documented to confer resistance. Indiana soybean [Glycine max (L.) Merr] fields infested with A. palmeri possessed diverse genotypes and herbicide surviving plants are likely to produce seed and spread if alternative control measures are not implemented.Herbicide-resistant weeds, especially Palmer amaranth ( Glycine max (L.) Merr.] cultivars resistant to glyphosate were released for commercial use , a species similar to A. palmeri   –21. A. pr states .A. tuberculatus, giant ragweed [Ambrosia trifida (L.)], and A. palmeri; however, some herbicide mixtures negatively affect herbicide efficacy , johnsongrass [Sorghum halepense (L.) Pers.], hemp sesbania [Sesbania herbacea (Mill.) McVaugh] or pitted morningglory [Ipomoea lacunosa (L.)] fresh weight at 4 WAT  control than 280 g ha-1 of fomesafen and 17 g ha-1 of cloransulam-methyl applied separately, respectively , A. tuberculatus, and A. trifida are problematic weeds Indiana growers contend with that have evolved resistance to glyphosate. A more alarming discovery was individuals were identified to harbor genes that allow A. palmeri survival to mixtures of chlorimuron-ethyl, fomesafen, and glyphosate. Although many A. palmeri in the non-treated check began to initiate inflorescence when the study was terminated at 21 DAT; plants that survived the three-way mixture had not begun to emerge inflorescence by experiment termination. Given the biology of the species, the potential for multiple HR plants to produce HR pollen and seed is likely in surviving plants allowed to continue growth in the field, creating a high risk for rapid multiple resistance evolution within individuals and populations. The obvious contribution of HR gene flow via seeds and pollen to the prevalence of multiple resistance highlights the importance of controlling weed escapes in the field, but also field borders, roadsides, ditch banks [ch banks .A. palmeri resistance evolution to fomesafen was reported in Arkansas [A. palmeri resistant to PPO-inhibiting herbicides has been reported in Indiana, Illinois, and Tennessee [A. palmeri biotype from Tennessee was not controlled with a POST fomesafen treatment and was also poorly controlled with soil-applied fomesafen and sulfentrazone treatments [PPX2L. Treating PPO-resistant A. palmeri with 1,500 g ha-1 of malathion followed by 263 g ha-1 of fomesafen 2 hours later reduced plant survival 22% more than fomesafen alone, indicating that fomesafen metabolism was responsible for A. palmeri survival [A. palmeri survival [A. palmeri.The first case of Arkansas . Since tennessee . Many PPeatments . Metabolsurvival . Revertisurvival ,45. Howesurvival . Future A. palmeri seed within Indiana is likely to occur, given that the weed is already present in the northern and southern regions. One study reported that A. palmeri adapted to Arkansas, Mississippi, Missouri, and Nebraska are able to develop and produce copious amounts of seed if introduced to Indiana [A. palmeri genotypes to documented HR mechanisms and confirmed A. palmeri survival to three-way herbicide mixtures. Management strategies that include cover crops, cultivation, hand-hoeing, planting G. max in narrow-rows, and selecting crop cultivars that rapidly canopy are necessary strategies that complement diversified PRE and POST herbicide programs.Continued spread of  Indiana . The datS1 Dataset(PDF)Click here for additional data file.S2 Dataset(PDF)Click here for additional data file.


Escherichia albertii is an emerging human enteropathogen. We report the draft genome sequence of E. albertii strain Mex-12/320a, isolated from an infant with diarrhea. The presence of the pathogenic island O122/IE6 and the nleA gene, previously found in diarrheagenic enteropathogenic Escherichia coli strains, suggests that E. albertii may cause acute diarrhea. Escherichia albertii is an emerging human enteropathogen. We report the draft genome sequence of E. albertii strain Mex-12/320a, isolated from an infant with diarrhea. The presence of the pathogenic island O122/IE6 and the nleA gene, previously found in diarrheagenic enteropathogenic Escherichia coli strains, suggests that E. albertii may cause acute diarrhea. Escherichia albertii is an emerging human enteropathogen which belongs to the attaching and effacing (A/E) family of gastrointestinal pathogens that include well-known human pathogens such as enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli , are significantly more prevalent among EPEC strains isolated from children with diarrhea than in those without  that appears to be specific to this species . The genome was assembled de novo using the SPAdes genome assembler v3.9.1 (http://cab.spbu.ru/software/spades/), and the accuracy of the genome assemblies was evaluated with REAPR 1.0.16 , generating 3,148,027 reads. Quality control, trimming, and filtering of raw sequencing data were performed with Trim Galore v0.4.1 , 5 are rRNA (rrn) operons, and 79 are tRNA genes. The genome sequence confirmed the presence of not only the LEE genes but also nleA and cdt-II  virulence genes previously found in other E. albertii strains  in an E. albertii strain.The draft chromosome sequence of  strains . To the E. albertii Mex-12/320a genome harbors virulence genes that have been previously reported in EPEC isolates from children with diarrhea. This finding adds to the growing body of evidence that E. albertii is an emerging pathogen of diarrheal disease  under the accession number SRR9024355.The draft genome assembly of