Candida has recently been suggested as a potential culprit for children’s dental caries, with evident maternal contributions. Therefore, this study aimed to obtain epidemiological data on the oral health and oral Candida carriage in a cohort of underserved US pregnant women, and reveal factors associated with their oral Candida carriage.Despite the well-documented associations between poor maternal oral health and increased risk for adverse birth outcomes and dental caries in children after birth, prenatal oral health care is under-utilized, especially among the underserved population. In addition, oral Candida species and Streptococcus mutans by culturing-dependent and -independent methods. Multiple logistic regression analyses were used to identify factors associated with oral Candida carriage and caries severity.Demographic-medical-oral hygiene practice data were collected. Comprehensive oral examination was conducted. Caries status and plaque index were recorded. Oral samples  were processed to identify p = 0.01). The average number of decayed teeth in pregnant and non-pregnant women was 3.9 and 3.1 (p > 0.05). Caries severity was positively associated with race (African American vs. white), plaque index and salivary Candida albicans level. C. albicans was the most predominant/abundant Candida strain, with cheek and tonsil as the most common colonized sites. The detection of C. albicans was 56%/56% in saliva and 40%/47% in plaque of the pregnant and non-pregnant groups, respectively. Study women’s oral Candida carriage is positively associated with hypertension , decayed teeth number  and salivary S. mutans level .Eighty-two socioeconomically disadvantaged women (48 pregnant and 34 non-pregnant) were enrolled. More pregnant women (79.1%) had > = 1 untreated decayed tooth when compared to their non-pregnant counterparts (47.1%)  will not only improve women’s oral health, but also present as a promising approach to reduce oral Candida carriage in women.Socioeconomically disadvantaged US women are in need of improved prenatal oral health, a large proportion of them have untreated decayed teeth and high carriage of oral  Poor maternal oral health is known to be associated with increased adverse birth outcomes, particularly preterm and low birth weight deliveries , 2. A sy< 0.001) . Further< 0.001) .Besides the association between maternal periodontal diseases and adverse birth outcomes, a strong correlation was also found between maternal carriage of dental caries related microorganisms and an increased risk for dental caries in children . Dental Streptococcus mutans and Lactobacillus species have traditionally been considered the prime microbial risk markers for dental caries [S. mutans and Lactobacilli is associated with nearly doubled children’s caries incidence versus low S. mutans and Lactobacilli mothers , after adjusting socio-demographics, feeding and care practices, and maternal dental status [Candida species in early childhood caries (ECC) and its synergistic interaction with S. mutans, has shed new light on potential fungus-focused approaches to early prediction and subsequent prevention of ECC. Candida species  have often been detected at higher levels in the oral cavity of children with ECC, compared to caries free children [C. albicans presented with > 5 times greater odds of experiencing ECC than children without this yeast strain [C. albicans carriage  [l caries –19. Chafchildren , and is children . In a ret strain . More imsamples) .Candida colonization was demonstrated in several studies. The vertical transmission rate from mother to newborns ranges from 14% typed by electrophoretic karyotyping and restriction endonuclease analysis of genomic DNA with pulsed-field gel electrophoresis, to 41% typed by DNA fingerprinting using a C. albicans strain-specific DNA probe [Candida strains [Maternal contribution to children’s NA probe , 31. We  strains .Even though the benefits of maintaining good maternal oral health during pregnancy are well demonstrated, many mothers-to-be do not receive timely prenatal oral care . A natioCandida carriage has been raised as a promising strategy in ECC prevention, understanding the factors associated with mother’s Candida carriage is essential to prevention of oral Candida colonization in infants. Therefore, this study was conducted to 1) obtain epidemiological data on oral health and oral carriage of Candida species in a group of US pregnant women with socioeconomic disadvantages, in comparison to their non-pregnant counterparts; 2) examine the relationship between demographic, medical and oral health status, and oral Candida carriage in pregnant women.Understanding the oral health condition of expectant mothers, particularly the ones with socioeconomic disadvantages can help us to design appropriate preventive and treatment strategies to help mothers gain better oral health during pregnancy, and provide further benefits to their children. Additionally, since controlling infants’ oral The study protocols were approved by the University of Rochester Research Subject Review Board (RSRB00056870 and RSRB00067191). All participants were informed of the study objectives and protocols, and gave written consent prior to study activities. Pregnant women were sampled from patients visiting the University of Rochester Highland Family Medicine (HFM) or Eastman Institute for Oral Health (EIOH). Control subjects, non-pregnant women, was a convenient sample collected from patients visiting the University of Rochester Eastman Institute for Oral Health. Both clinics, HFM and EIOH, serve a large body of low-income patients, with a range of 40% African Americans, 40% White and 20% others. The status of holding New York state-support medical insurance was used as a filter for the selection of women with socioeconomic disadvantages. The age and socioeconomic status of the subjects were used to match between the pregnant and non-pregnant groups.We calculated the sample size based on the estimation that 60% of pregnant women have untreated decayed teeth, compared to 21.8–30.3% of women in the general population who have untreated decayed teeth, reported by CDC . The aveFor the pregnant women group, individual who met for the following inclusion criteria and exclusion criteria were enrolled. For the control group women who are not pregnant, all inclusion and exclusion criteria apply except the items relate to pregnancy. Inclusion criteria included:1) Female, equal or older than 18 years of age. 2) Pregnant with singleton fetus and in her 3rd trimester (time period extending from the 28th week of gestation until delivery). 3) Eligible for New York state-supported medical insurance, which is determined by income level .Exclusion criteria included:1) Subjects who have decisional impairment deeming incapable of making an informed decision about her participation in the study. 2) Subjects who received oral and/or systemic antifungal therapy within 90 days of the baseline study visit. 3) Subjects who have severe systemic medical conditions  that make them prone to yeast infections.Data on demographics were self-reported by the study subjects. Data on the medical background and medications were collected through self-reporting and confirmed by electronic medical records. See Additional file Candida infection site, for pregnant women with positive detection of salivary or plaque C. albicans, additional mucosal swab samples were collected from cheek mucosa, labial mucosa, dorsal surface of tongue, hard palate, and tonsils using FlocqSwabs  at the next study visit.The whole non-stimulated saliva samples were collected by spitting into a sterilized 50 ml centrifuge tube. Approximately 1 ml of saliva was collected for each subject. Supragingival plaque from the whole dentition  was collected using a sterilized periodontal scaler . The plaC. albicans test was prescribed for 33 pregnant women by their physicians within a window of 12 months before entering our study; available vaginal C. albicans data was obtained through the electronic medical records.Vaginal C. albicans by incubating at 37 °C for 48 h. This medium permits presumptive identification of several clinically important Candida species including C. albicans, C. krusei, C. galabrata, C. dubliniensis, etc., based on the colony color and morphology [C. albicans detection sensitivity (98.6%) and specificity (98.8%) [S. mutans was isolated using Mitis Salivarius with Bacitracin selective medium by incubating at 37 °C for 48 h and identified by colony morphology [Candida spp. and S. mutans on each plate were counted and recorded as colony forming unit (CFU). Additionally, C. albicans and S. mutans were further identified using colony polymerase chain reaction method. The probes used for C. albicans were forward primer 5’CGATTCAGGGGAGGTAGTGAC3’ and reverse primer 5’GGTTCGCCATAAATGGCTACCAG 3′. The probes used for S. mutans were forward primer 5′ TCGCGAAAAAGATAAACAAACA 3′ and reverse primer 5′ GCCCCTTCACAGTTGGTTAG 3′ [After the sample collection, the clinical samples  were stored on ice and transferred to the lab located at the University of Rochester Center for Oral Biology within 2 h for laboratory testing. The saliva and plaque sample were gently vortexed and sonicated to break down the aggregation before plating. The sonication cycle was repeated three times, with 10 sec sonication and 30 sec rest on ice. BBL™ CHROMagar™ Candida  was used to isolate rphology . CHROMag (98.8%) . S. mutarphology . ColonieGTTAG 3′ .Candida status. The characteristics of the two groups  were compared using t-test for continuous data and Chi-square or Fisher’s exact tests for categorical data. One-way ANOVA and Turkey test was used to compare the C. albicans carriage between swab samples taken from different mucosal sites. Mann-Whitney U Test was performed to compare the difference of decayed teeth number in relation to other binary variables including pregnancy, smoking, inhaler use for asthma, and ethnicity (Hispanic and non-Hispanic). Spearman’s rank was used to measure the correlation between variables . Cohen’s kappa was calculated to test the agreement between oral and vaginal C. albicans detection. A cumulative logistic regression analysis was used to test variables associated with caries severity among study subjects. The caries severity was grouped into three levels: 0 (no decay), 1 (<=3 decayed teeth) and 2 (> 3 decayed teeth). Multiple logistic regression analyses were used to identify predictors of Candida carriage among study subjects. Estimated odds ratios (OR) and 95% confidence intervals (CIs) were calculated for the variables that were statistically significant. The variables in the regression analyses were selected in multiple steps. We first selected a list of variables from the dental literature which might be potentially correlated with the outcome variables. Then, for the covariates like age, race, tooth brushing frequency, even though they have high p-values and are insignificant in the model, we still retained them in the model. Other variables, especially clinical characteristics, such as diabetes, asthma, hypertension, were selected based on p-values. We used a cut-off of 0.2 for the inclusion of those variables. All statistical tests were two-sided with a significance level of 5%. SAS were used for all statistical analyses.During data analysis, women in the study were grouped based on their pregnancy status and oral C. albicans carriage (p > 0.05). Pregnant women’s plaque index and salivary S. mutans carriage was higher than non-pregnant women’s (p < 0.05). Strikingly, 79.1% of pregnant women and 47.1% of non-pregnant women had at least 1 untreated decayed tooth (p = 0.01); the average number of decayed teeth (DT) of the study pregnant women and non-pregnant women are 3.9 ± 3.8 and 3.1 ± 4.8; no difference regarding the decayed teeth number was seen between pregnant and non-pregnant groups (p > 0.05). Moreover, the average number of DMFT of the study pregnant women and non-pregnant women are 6.9 ± 6.4 and 7.5 ± 4.5 (p = 0.09).A total of 48 eligible pregnant women and 34 non-pregnant women were enrolled in this study. None of the expectant mothers reported having dental exams since their pregnancy. Characteristics of the study population are shown in Table Candida carriage is shown in Fig. Candida detection rates were similar: 56 and 56% in saliva, and 40 and 47% in plaque, in the pregnant and non-pregnant groups, respectively. C. albicans was the most predominant and abundant species found in Candida carriers of both groups , tongue (42%), hard palate (29%) and inner side of the upper and lower lips (25%) , shown in Fig. p > 0.05).Among 24 pregnant women who had positive C. albicans detection among these 33 pregnant women  showed a slight agreement between oral and vaginal C. albicans detection.A comparison was made between oral and vaginal p = 0.026) and inhaler use for asthma (p = 0.047); The decayed teeth number was marginally different between pregnant and non-pregnant groups (p = 0.051); no difference were detected between smoking and non-smoking groups (p = 0.27). The correlation between variables tested by Spearman’s rank is shown in Table S. mutans level, and salivary and plaque C. albicans level. To analyze factors associated with caries severity in socioeconomically disadvantaged women, a cumulative logistic regression model was used. The factors input into the analysis were age, race, ethnicity, pregnancy, smoking, tooth brushing frequency, plaque index, salivary Candida level and salivary S. mutans level. Among all the factors, caries severity was positively associated with race , plaque index  and salivary C. albicans carriage , detailed in Table The decayed teeth number significantly differed between these groups: ethnicity p = 0.06 and inhCandida carriage in socioeconomically disadvantaged women, we further grouped all study women (both pregnant and non-pregnant) into two groups based on the C. albicans status - positive and negative groups. Characteristics of the study population in two groups are shown in Table p > 0.05) between C. albicans positive and C. albicans negative group in terms of age, race, ethnicity, medical background, tooth brushing habit, and plaque index. More smokers were found in women with positive oral C. albicans detection (p = 0.04). Furthermore, higher percentage of having untreated decayed teeth, higher number of decayed teeth, higher number of decayed/missing/filled teeth, and higher carriage of salivary S. mutans were all found in the women with positive C. albicans detection (p < 0.05).To analyze the factors associated with oral C. albicans in women with socioeconomic disadvantages was significantly associated with hypertension condition , higher decayed teeth  and higher salivary S. mutans level . Other demographic, medical background, and oral hygiene practice characteristics were not associated with the prevalence of oral C. albicans in women with socioeconomic disadvantages, including pregnancy status.Through multivariate logistic regression analysis  [Candida carriage was 1.4 × 103 CFU/ml in pregnant women. Intriguingly, although none of the pregnant women presented clinical manifestation of oral candidiasis, more than 50% of them could be diagnosed as oral candidiasis based on the salivary Candida CFU established by Epstein et al [Candida detection site in the oral cavity, a combination of gauging and swishing oral antifungal rinse might provide better outcomes in treating oral candidiasis.Second, we revealed a high oral 16–49%) , 49. The% , 49. Tin et al , 400 CFUCandida, we found that pregnancy status is not significantly associated with Candida detection among the study population. This resonates with a previous study, in which pregnancy and diabetes independently did not influence the prevalence of fungi in the oral cavity and rectum of pregnant women [Candida infections [Third, upon examining the association between pregnancy and oral nt women . While sfections , 52. It Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in the subgingival plaque, were significantly higher during the early and middle stages of pregnancy, compared to the non-pregnant group [A. actinomycetemcomitans and Candida in the second and third trimesters of pregnancy compared to non-pregnant women [Besides the unclear mechanistic effect of pregnancy on the oral microbiota, the association between pregnancy and vaginal/oral microorganism carriage has revoked various discussions. For instance, one study revealed that oral microorganisms change during different stages of pregnancy. When comparing the abundance of seven common bacterial species in the oral cavity of non-pregnant women, early pregnancy, mid-pregnancy, and late pregnancy, the total viable microbial counts in all stages of pregnancy were higher than those of the non-pregnant women, especially in early pregnancy , and levnt group . These rnt women .S. mutans level are associated with oral Candida carriage in women, which have not been reported by previous studies. In recent years, with more research endeavors drawn to the association between oral Candida and tooth decay in children, research has indicated that children’s oral Candida carriage is associated with S. mutans level, and decayed teeth number. The association between Candida and dental caries identified in the children’s population might explain our findings in the adult population. This finding indeed intrigues an important clinical indication, which is restoring decayed teeth in mothers during pregnancy might offer therapeutic potential for controlling oral Candida in the mothers, preventing oral Candida colonization in infants by reducing the vertical transmission, and subsequently preventing dental caries in children.Fourth, we found that hypertension, decayed teeth number, and salivary C. albicans detection and indicated a high correlation, which is similar to some other study findings. Lockhart et al. [C. albicans, between oral and vaginal habitats, which implies that the effective oral/vaginal Candida control strategy demands the consideration of both oral and vaginal origins.Lastly, we examined the association between oral and vaginal t et al.  showed tCandida carriage of the pregnant women to their non-pregnant counterparts. Although we observed that oral Candida detection is independent of the pregnancy, the results need to be confirmed by ideal study designs that examine all stages of pregnancy, including pre-, during, and post-pregnancy. 2) Limited sample size may have compromised the power of our multiple regression analyses. 3) The study was conducted in a single US city. Due to the nature of the single-site conducted study and small convenient sample size, the study results cannot be generalized to other populations. 4) The non-pregnant group was a convenience sample. The pregnant and non-pregnant subjects were enrolled from clinics where a majority of the patients are low-income individuals. The age and socioeconomic status were used to match the pregnant and non-pregnant groups. The status of holding New York state-support medical insurance was used as a filter for the selection of women with low socioeconomic status. The eligibility for state-support insurance is determined by income level . Although we did not conduct a case-by-case match for the education background, race, and ethnicity, there is no statistical difference in regard to the distribution of different races (p = 0.8) and ethnicity (p = 0.91) between the two groups (see Table The following limitations need to be considered when interpreting our study results: 1) With a cross-sectional study design, we only observed one time-point (the 3rd trimester) during the pregnancy and compared the oral condition and Candida. Study women’s oral Candida carriage is positively associated with hypertension, decayed teeth number, and salivary S. mutans level. Due to the observed significant association between the decayed teeth number (caries severity) and oral Candida carriage, providing oral health care during pregnancy (including limit decayed teeth) will not only improve women’s oral health, but also present as a promising approach to reduce oral Candida carriage in women, and subsequently benefit their offspring.Socioeconomically disadvantaged US women are in need of improved prenatal oral health, a large proportion of them have untreated decayed teeth and high prevalence and carriage of oral Additional file 1. Demographic and medical backgroundAdditional file 2: Figure S1. Oral Candida status in non-pregnant mothers. (A) Oral  Candida species detection in non-pregnant women. (B). Candida species carriage in the saliva and plaque of non-pregnant women.


Ixodes scapularis, is the primary vector of the Lyme disease spirochete Borrelia burgdorferi in North America. Though the tick is found across the eastern United States, Lyme disease is endemic to the northeast and upper midwest and rare or absent in the southern portion of the vector’s range. In an effort to better understand the tick microbiome from diverse geographic and climatic regions, we analysed the bacterial community of 115 I. scapularis adults collected from vegetation in Texas and Massachusetts, representing extreme ends of the vector’s range, by massively parallel sequencing of the 16S V4 rRNA gene. In addition, 7 female I. scapularis collected from dogs in Texas were included in the study.The blacklegged tick, I. scapularis ticks had a more diverse bacterial microbiome in comparison to the female ticks. Rickettsia spp. dominated the microbiomes of field-collected female I. scapularis from both regions, as well as half of the males from Texas. In addition, the male and female ticks captured from Massachusetts contained high proportions of the pathogens Anaplasma and Borrelia, as well as the arthropod endosymbiont Wolbachia. None of these were found in libraries generated from ticks collected in Texas. Pseudomonas, Acinetobacter and Mycobacterium were significantly differently abundant (p < 0.05) between the male ticks from Massachusetts and Texas. Anaplasma and Borrelia were found in 15 and 63% of the 62 Massachusetts ticks, respectively, with a co-infection rate of 11%. Female ticks collected from Texas dogs were particularly diverse, and contained several genera including Rickettsia, Pseudomonas, Bradyrhizobium, Sediminibacterium, and Ralstonia.Male I. scapularis ticks vary by sex and geography, with significantly more diversity in male microbiomes compared to females. We found that sex plays a larger role than geography in shaping the composition/diversity of the I. scapularis microbiome, but that geography affects what additional taxa are represented (beyond Rickettsia) and whether pathogens are found. Furthermore, recent feeding may have a role in shaping the tick microbiome, as evident from a more complex bacterial community in female ticks from dogs compared to the wild-caught questing females. These findings may provide further insight into the differences in the ability of the ticks to acquire, maintain and transmit pathogens. Future studies on possible causes and consequences of these differences will shed additional light on tick microbiome biology and vector competence.Our results indicate that the bacterial microbiomes of The online version of this article (10.1186/s12866-019-1514-7) contains supplementary material, which is available to authorized users. Ixodes scapularis, is the major vector of the Lyme disease agent, Borrelia burgdorferi, in North America [Ticks transmit a greater variety of human and animal pathogens than any other disease vector and have significant public health impacts worldwide –7. The b America . This ti America –16.I. scapularis across the eastern United States, Lyme disease cases are concentrated in the northeastern and upper midwestern states, whereas the disease is very rare or absent in the southern portion of the vector’s range [B. burgdorferi among I. scapularis in the northeastern U.S. has been reported to be as high as 30–50% [I. scapularis in the northeastern and midwestern regions [With more than 30,000 reported cases per year and an estimated 10-fold greater burden than the reported case counts, Lyme disease is the most common vector-borne illness in the U.S. –19. Desps 30–50% –22, whils 30–50% –25. Over regions .B. burgdorferi-harboring ticks, availability of the B. burgdorferi competent hosts, tick-behavior, seasonal activity of the ticks, and environmental variables influence the risk of Lyme disease [I. scapularis can be encountered by humans during warm season of the year. But, in the southeastern U.S., human encounter occurs primarily with the adults I. scapularis ticks as immature ticks rarely seeks hosts in the region [I. scapularis ticks has increasingly been studied in recent years [Ixodes ticks varies with sex, species and geography [I. scapularis ticks from eastern and southern Ontario do not differ significantly with regard to geographic origin, sex or life stages [I. scapularis can be influenced by the environmental temperature [I. scapularis adults collected from natural vegetation from Texas and Massachusetts, representing opposing ends of the vector’s range and possessing distinct climates, by sequencing the hypervariable region 4 (V4) of the 16S ribosomal RNA (rRNA) gene using an Illumina MiSeq platform. Adult ticks were chosen to provide a fair comparison of the tick microbiomes from two regions with different geography and climate, and to provide ample DNA per sample without the need for pooling of multiple, smaller life stages.Many factors, including the density of host-seeking  disease , 27–33, e region . Recent e region , 35–39. nt years –43. In teography . By conte stages . These cperature . With thI. pacificus [I. scapularis, with potential consequences for vector competence. To investigate how host blood meal affects microbiome, we also analysed the bacterial microbiomes of dog-fed female I. scapularis ticks during this study.Host blood meal has been shown to affect microbial diversity in acificus , a closeI. scapularis samples  prevalence in males and 100% (99.9–100%) prevalence in females. The prevalence of Proteobacteria in female ticks collected from dogs in Texas was 93% (86–100%). Proteobacteria were prevalent at 84.8% across ticks from Massachusetts, with 71.9% (0–99.9%) prevalence in males and 98.5% (11.1–88.9%) in females. Other common phyla in the microbiomes of the Texas ticks  included various proportions of Actinobacteria, Bacteroidetes, and Firmicutes. In addition to these non-proteobacterial phyla found in the Texas ticks, Massachusetts ticks were represented by the Spirochaetes, albeit with higher abundance in males (63% of them with ≥1% relative abundance) compared to that of the females (37%)  had a more complex microbiome comprised of Mycobacterium (mean = 23%), Acinetobacter (22%), Sphingomonas (5%), Methylobacterium (4%), Corynebacterium (2%), Staphylococcus (2%), Escherichia (2%), Rickettsia (2%), Sphingobium (2%), Rhizobium (1%), Pseudomonas (1%), and others. There was the lack of variation in microbiome composition of field-collected Texas ticks between the collection years , followed by Borrelia , Wolbachia (1%), Anaplasma (0.1%), and others  and 1% (2–79%), respectively. Wolbachia, with a mean relative abundance of 1.1% (0–27%), was found in more than 25% of the females from the region. Microbiomes of male ticks collected in Massachusetts were represented by multiple genera, including Pseudomonas , Borrelia (23%), Rickettsia (20%), Anaplasma (9%), Bradyrhizobium (5%) Wolbachia (4%), Ralstonia (4%), Acinetobacter (1%), and others. Altogether, 17 genera and 7 additional unclassified taxa  had 1% or greater abundance in at least one of the 32 libraries generated from male ticks collected in Massachusetts. Borrelia  was found in 66% of the Massachusetts males, while Anaplasma  was detected in about 19% of the males. Moreover, Wolbachia was also found in about 28% of the male ticks from Massachusetts.Libraries generated from ticks collected in woodlands in Massachusetts showed a similar dependence on the sex of the host. Pseudomonas , Acinetobacter (p = 0.006) and Mycobacterium (p = 0.004). Additionally, Anaplasma, Borrelia and Wolbachia bacteria were found in both male and female ticks from MA, but not from those in TX.Significant differences were found in the mean relative abundance of certain genera in males from Texas and Massachusetts including, Rickettsia (mean abundance = 53%), but the abundances of Pseudomonas (20%), Bradyrhizobium (7%), Sediminibacterium (5%), Ralstonia (4%), and Acinetobacter (2%) were much higher than in the microbiomes of questing ticks collected from foliage  gut microbiome of the female ticks, as described in Thapa et al. (2018) [in-silico removal of Rickettsia sequences from the female dataset, none of the field-collected female samples from Texas had sufficient sequences remaining to pass the inclusion criteria (as described in the beginning of the results section) needed to proceed for further analysis. Five female samples from Massachusetts also did not meet the inclusion criteria for further analysis after removing Rickettsia. Of the remaining 25 female samples, all collected from Massachusetts, only 13 had more than 1000 reads  after deleting Rickettsia sequences. In-silico removal of Rickettsia from the Massachusetts female datasets revealed the previously hidden bacterial composition . In contrast to a very low distribution of Borrelia in their full profiles , the relative abundance of Borrelia in 11 Rickettsia-deleted female samples  ranged from 45 to 100% (mean = 79%)  . After i018 . Aftp < 0.0001). However, female ticks collected from dogs in Texas had a significantly higher number of observed OTUs in comparison to the foliage-associated female ticks from either Texas or Massachusetts    was performed to normalize the number of sequences in each sample . Regardlsee Fig. . Similarly) Fig. , and femI. scapularis ticks collected from Massachusetts clustered separately from others, as did half the males from Texas in an unweighted PCoA plot of axis-1 vs axis-2. PCoA of unweighted UniFrac distances of bacterial communities showed that the first two axes (PCo1 and PCo2) explained 10.5 and 3.7% of the variation in the data, respectively . The plot also demonstrated that the male samples from TX clustered separately when compared to the males from Massachusetts (p = 0.001), except one outlier from MA within the cluster of TX males. Female ticks collected from dogs in Texas formed a coherent cluster close to the field-captured males from Massachusetts. No clear clustering was observed in a PCoA plot of the weighted UniFrac distance metrics based on the collection site or sex of the tick  field-collected I. scapularis from Massachusetts  and female (p = 0.26). . In terms of female ticks, Rickettsia was almost exclusively dominant in both regions, but Massachusetts females contained Borrelia, Anaplasma, and Wolbachia, while these bacteria were absent from the ticks collected in Texas.Although the distribution of individual bacterial taxa varied among male and female ticks from Massachusetts, there was no significant difference in bacterial composition between I. scapularis ticks from our previous publication [p < 0.05 for all comparisons). In female ticks, Proteobacteria dominated the microbiome of both colony-reared and field-collected ticks (questing and dog fed) . Bacterial diversity in colony-reared male ticks found to differ significantly from that of the wild-caught males from TX or MA (unweighted UniFrac PERMANOVA p = 0.001 for both comparisons). The colony-reared female ticks also differed significantly in beta diversity (as measured by unweighted UniFrac distances) when compared to that of the female ticks collected from vegetation in TX and MA (p = 0.001). However, the colony-reared I. scapularis females were no more diverse than the female ticks collected from dogs (p = 0.06).We also compared the baseline microbiome data of the colony-reared lication  to that I. scapularis ticks from both Texas and Massachusetts agrees with a previous study [Rickettsia in all field-collected female ticks from Texas, and a very high dominance (97%) in females from Massachusetts, is consistent with previous reports for the microbiome of I. scapularis [Rickettsia in larvae and nymphal I. scapularis [The dominance of Proteobacteria in us study  of wild-us study . Our finapularis –44 and hapularis , 22.Rickettsia likely reflect a mutualism between this endosymbiont and the host, and most likely belong to the endosymbiont Rickettsia buchneri [R. buchneri has been shown to provide a source of vitamins to the tick [Rickettsia also contains many potentially pathogenic species, including Rickettsia rickettsii, R. japonica, R. akari [R. parkeri [, but these bacteria are not known to be vectored by I. scapularis. By contrast, high prevalence of R. buchneri endosymbionts in female ticks is generally associated with the ovaries [Rickettsia was also highly prevalent in male ticks, our findings suggest that Rickettsia resides in other body parts of the male ticks. This is consistent with previous reports of R. parkeri detected in male tissues of Amblyomma maculatum [The high numbers of buchneri . R. buchthe tick . The genR. akari  and R. p parkeri , but the ovaries , 51. As aculatum .Pseudomonas (MA = 23% vs TX = 1%), Acinetobacter (MA = 1% vs TX =22%), Mycobacterium (MA = 0.4% vs TX = 23%) and the exclusivity of Borrelia and Anaplasma to MA, suggests a geographical and/or ecological variation of the microbiota in these ticks with public health consequences. Our findings of slightly higher abundance of Borrelia or Anaplasma in male ticks from Massachusetts compared to the female ticks suggest the possible roles of the underlying microbial community in the male ticks for pathogen acquisition. However, it should also be noted that some differences between males and females may be artifacts arising from differences in sequencing depth between males and rickettsia-subtracted females. Caution is therefore warranted in interpretation of these differences. In addition, the large variation between microbiomes of wild-caught Texas male ticks suggests the possibility of two distinct microbiomes. However, all the ticks were collected from the same habitat in Texas during 2016 and 2017, and the pattern is similar for both collection years. The variations between the microbiomes of Texas male ticks might have to do with prior host blood meal. The mean abundance of Borrelia, which could include pathogenic B. burgdorferi and B. miyamotoi¸ was higher in males (35%) in comparison to the females (< 2%) and in the range of previous studies [Borrelia and Anaplasma detected in I. scapularis ticks collected from Massachusetts. In comparison to the traditional PCR-based approaches used previously [Borrelia 16S rRNA gene sequences are due to B. burgdorferi (the causative agent of Lyme disease), but could be partially due to B. miyamotoi (relapsing fever group bacterium). B. miyamotoi has also been identified in this area, albeit at substantially lower numbers, with 2.3% of ticks tested from Cape Cod in 2016 found positive for this bacterium [I. scapularis samples yielding Borrelia reads from 16S rRNA Illumina sequencing produce amplicons in PCR testing of the B. burgdorferi specific ospC gene [A. americanum tick [Borrelia in I. scapularis ticks collected from North Truro in Cape Cod, Massachusetts is in line with the findings of Xu et al. (2016) [I. scapularis ticks tested from Nantucket county in Massachusetts were B. burgdorferi positive, and also consistent with unpublished work conducted by our laboratory using nested PCR methods (data not shown).The complexity of the microbiomes of male ticks collected in both Massachusetts and Texas may reflect acquisition from the environment, as relatives of many genera found in the guts of male ticks are considered free-living (not host-associated) bacteria. Differences, such as the relative abundance of  studies , 44. Xu acterium . Furthernum tick . HoweverAnaplasma-Borrelia co-infection rate of 11% in the ticks from Massachusetts we report was substantially higher than a previous study [I. scapularis from Massachusetts, where 1.8% of the ticks were coinfected by B. burgdorferi and A. phagocytophilum. The higher rate of co-infection in this study could be attributed to the overall higher prevalence of Borrelia.The us study  on humanWolbachia in more than 25% of the ticks from Massachusetts was not expected. Wolbachia are known to exhibit endosymbiotic mutualism with insects [I. scapularis. Although Wolbachia has been known to induce resistance to dengue virus when introduced into Aedes aegypti mosquitoes [Wolbachia in the I. ricinus tick, a major European vector of the Lyme disease agent, is due to the presence of the endoparasitoid wasp Ixodiphagus hookeri, and not representative of a true endosymbiont of the tick [Wolbachia reported here is likely not a true mutualism with I. scapularis, but rather may indicate the presence of an unidentified parasite.The detection of  insects , 56, and insects , 58, butsquitoes , Plantarthe tick . Thus, tRickettsia in female ticks. Furthermore, a significantly higher Shannon diversity in male ticks suggests that the community of male ticks were more diverse as well as even, compared to the females.The higher bacterial richness in the microbiome of male ticks compared to female ticks, regardless of the geographical origin reflects dominance of I. scapularis ticks collected from dogs in Texas, in comparison to the wild-caught females from both states, suggests that the tick microbiome may shift as the result of a recent blood meal. The microbiomes of the female ticks that originated from dogs in Texas closely matches that of the male ticks from Massachusetts in terms of diversity but not in community membership, further supporting the idea that the bacterial microbiomes of female I. scapularis ticks vary with their sample source. One possible explanation could be that recent blood feeding led to increased abundance of midgut bacteria, lessening the overall relative impact of the rickettsial endosymbiont on subsequent analyses.The complexity of libraries generated from female I. scapularis ticks in comparison to the wild-caught ticks could be attributed to multiple factors, including the type of previous blood meal, and the environmental/ecological parameters. The difference in microbiomes of I. scapularis ticks from Massachusetts and Texas, including the ticks fed on dogs in Texas may, also reflect the seasonal effects on the tick microbiome. Indeed, we have previously shown that the environmental temperature can influence the endogenous tick microbial community composition in colony-reared I. scapularis [Difference in the composition and diversity of the microbiome of colony-reared apularis .I. scapularis ticks from Texas and Massachusetts demonstrated that the bacterial microbiota of the ticks varies by sex and geographic origin. The main findings of this study are that sex plays a larger role than geography in shaping the composition/diversity of the I. scapularis microbiome, but that geography affects what additional taxa are represented (beyond Rickettsia) and whether pathogens are found. In addition, the microbiome of dog-fed female I. scapularis ticks is more complex than those of the wild-caught females.Analyses of the microbiomes of field-collected adult Taken together, our findings may provide further insight into the sexual and regional differences in the ability of the ticks to acquire, maintain and transmit pathogens. Future studies on functional and mechanistic aspects of the tick microbiome, including possible causes  and consequences of these differences will help us better understand the microbiome biology of the ticks and vector competence. These efforts may ultimately aid development of strategies to control the risk and transmission of tick-borne diseases.I. scapularis were collected in the Davy Crockett National Forest near Kennard, Texas and from the North Truro area in the Barnstable county of Cape Cod, Massachusetts. Due to the difference in activity levels of ticks in different local environments, sample collection in Massachusetts was done during the late spring while ticks from Texas were collected during autumn. I. scapularis is endemic to both Cape Cod, located in the northeast U.S. [2 piece of white cloth attached to a pole gently over and around the vegetation where ticks were likely to be present. All encountered ticks were collected with fine-tipped tweezers and placed into sterile collection vials containing cotton fabric for housing. Ticks were categorized by location (TX or MA) and sex . All ticks were then preserved at ˗20 °C until DNA extraction. In addition, seven I. scapularis females collected from dogs (pulled off with tweezers) in North Texas were included in the study. These dog-fed ticks were unengorged to partially engorged. Details of the collection sites and dates are provided in Table 2During 2016 and 2017, a total of 115 adult ast U.S. , and Triast U.S. . StandarAll tick samples were treated in sequence with 10% sodium hypochlorite and molecular biology grade water to reduce surface contamination. Sterilization techniques using sodium hypochlorite solution have previously been demonstrated to significantly eliminate the bacteria and DNA on the tick surface . Each whEach DNA extract was first assessed by PCR to amplify the tick mitochondrial 16S rRNA gene as a sample positive control, as previously described  using 164, 0.5-μl 10 μM forward primer, 0.5-μl 10 μM reverse primer, 0.1-μl of 5 U/μl Accuprime™ Taq DNA Polymerase High Fidelity, 10-μl (43–554 ng) of template DNA and 7.9-μl molecular biology grade water. PCR was carried out in a BioRad C1000 Touch™ thermal cycler with the following cycling parameters: an initial denaturation at 94 °C for 2 min followed by 30 cycles  consisting of denaturation at 94 °C for 30 s, annealing at 55 °C for 40 s, and extension at 68 °C for 40 s, with a final extension at 68 °C for 5 min and a 4 °C indefinite hold. Amplicon quality was evaluated by visualizing under UV light after separation in 1.5% agarose gel after electrophoresis. No template negative controls were used during the PCR runs.DNA was amplified in duplicates by PCR using 515F/806R primers that target the hypervariable region four (V4) of the bacterial 16S rRNA gene. The primer set (forward: 5′-GTGCCAGCMGCCGCGGTAA-3′ and reverse 5′-GGACTACHVGGGTWTCTAAT-3′) had overhanging Illumina sequencing adaptors. Earth Microbiome Project (EMP) 16S Illumina Amplification Protocol was followed  with minTaq DNA Polymerase High Fidelity (5 U/μl), 5-μl PCR product and 29.8-μl molecular biology grade water. Amplification was performed in a BioRad C1000 Touch™ thermocycler with the following program: 94 °C for 3 min followed by 8 cycles of 94 °C for 30 s, 55 °C for 30 s, 68 °C for 30 s, and 68 °C for 5 min, followed by holding at 4 °C. The final purified PCR products were quantified using a Qubit® 2.0 fluorometer  and libraries were pooled in equimolar amounts. The pooled library  was loaded in the MiSeq Reagent Kit v2  and paired-end (2 × 250) sequencing (500 cycles) was performed on an Illumina MiSeq instrument per manufacturer’s recommendations. Two negative controls (a blank extraction control and a no-template PCR negative control) were sequenced alongside of the samples to monitor for background contamination. A 5% PhiX DNA was also included in the run as an internal control.PCR amplicons in duplicate sets were combined for each sample. Purification of the PCR products were performed using AMPure XP magnetic beads, and 16S libraries for a total of 122 samples were prepared following the Illumina 16S metagenomic sequencing library preparation protocol with the following minor modifications: the reaction mixture for index PCR (per 50 μl reaction) consisted of 5-μl 10X Accuprime™ PCR Buffer II, 5-μl Nextera XT Index Primer 1, 5-μl Nextera XT Index Primer 2, 0.2-μl Accuprime™ Data were analyzed in the Windows version of Microsoft Excel 2016 , XLSTAT-Ecology , and R software (version 3.4.1) as well as in mothur software . GraphPaRickettsia sequences were removed from the dataset using the remove.lineage command in mothur, as described in Thapa et al. (2018) [. (2018) . Relativ. (2018)  in the d. (2018) , which t. (2018) , was app. (2018) . The levAdditional file 1:Figure S1. Rarefaction curves of the number of OTUs observed in male and female I. scapularis.Figure S2. Relative abundance of bacterial phyla in I. scapularis ticks from Texas and Massachusetts, USA. Figure S3. Bacterial richness (ACE and Chao1 estimators) in I. scapularis ticks. Figure S4. PCoA plot of weighted UniFrac distance metrics in male and female I. scapularis ticks collected from Texas and Massachusetts, USA. Figure S5. Unweighted PCoA plot of Borrelia-positive and Borrelia-negative I. scapularis males and females collected from Massachusetts, USA. Figure S6. Comparison of the bacterial microbiomes in colony-reared and field-collected I. scapularis ticks. (DOCX 1633 kb)


Glyphosate-based herbicides (GBHs) are broad-spectrum herbicides that act on the shikimate pathway in bacteria, fungi, and plants. The possible effects of GBHs on human health are the subject of an intense public debate for both its potential carcinogenic and non-carcinogenic effects, including potential effects on the endocrine system The present pilot study examine whether exposure to GBHs at the dose of glyphosate considered to be “safe” (the US Acceptable Daily Intake - ADI - of 1.75 mg/kg bw/day), starting from in utero life, affect the development and endocrine system across different life stages in Sprague Dawley (SD) rats.Glyphosate alone and Roundup Bioflow, a commercial brand of GBHs, were administered in drinking water at 1.75 mg/kg bw/day to F0 dams starting from the gestational day (GD) 6 (in utero) up to postnatal day (PND) 120. After weaning, offspring were randomly distributed in two cohorts: 8 M + 8F/group animals belonging to the 6-week cohort were sacrificed after puberty at PND 73 ± 2; 10 M + 10F/group animals belonging to the 13-week cohort were sacrificed at adulthood at PND 125 ± 2. Effects of glyphosate or Roundup exposure were assessed on developmental landmarks and sexual characteristics of pups.In pups, anogenital distance (AGD) at PND 4 was statistically significantly increased both in Roundup-treated males and females and in glyphosate-treated males. Age at first estrous (FE) was significantly delayed in the Roundup-exposed group and serum testosterone concentration significantly increased in Roundup-treated female offspring from the 13-week cohort compared to control animals. A statistically significant increase in plasma TSH concentration was observed in glyphosate-treated males compared with control animals as well as a statistically significant decrease in DHT and increase in BDNF in Roundup-treated males. Hormonal status imbalances were more pronounced in Roundup-treated rats after prolonged exposure.The present pilot study demonstrate that GBHs exposure, from prenatal period to adulthood, induced endocrine effects and altered reproductive developmental parameters in male and female SD rats. In particular, it was associated with androgen-like effects, including a statistically significant increase of AGDs in both males and females, delay of FE and increased testosterone in female.The online version of this article (10.1186/s12940-019-0453-y) contains supplementary material, which is available to authorized users. Glyphosate [IUPAC chemical name N-(phosphonomethyl)glycine] is the active ingredient of all glyphosate-basedherbicides (GBHs), which is the most widely applied pesticide worldwide including the commercial formulation “Roundup” , 31. Sinw/v) polyoxyethyleneamine (surfactant) [In vivo, sexual development is controlled by hormones and is therefore highly sensitive to exogenous substances with endocrine-related effects. In rats, different studies have investigated the effects of high doses of Roundup administered to rats prenatally and postnatally on sexual maturity. A range of significant effects were observed, including i) both increased or reduced concentration of total testosterone (TT) in males treated with Roundup formulation (Monsanto of Brazil) containing 18% (factant) , 41; ii)factant) . Similarfactant) . Finallyfactant) .Pure glyphosate might be less potent than GBHs (such as Roundup formulations) in terms of reproductive toxicity. Testicular toxicity and reduced sperm counts, but no hormone variations, were observed in sexually mature male Sprague-Dawley (SD) rats treated active ingredient glyphosate, only at the highest dose level of 500 mg/kg bw/day .In addition, evaluation of glyphosate and GBHs by international agencies is not without controversies. No evidence of interaction of glyphosate with the estrogen pathway was detected in the Endocrine Disruptor Screening Program (EDSP) conducted by the US Environmental Protection Agency (EPA) . HoweverBecause to date relatively few human health studies have been conducted, the epidemiological evidence of GBH effects on reproductive and developmental health outcomes is too limited to draw conclusions. The Ontario Farm Family Health Study (OFFHS) showed a significant association between preconception exposure to pesticide products containing glyphosate and increased risk of spontaneous abortion , 43. A rTaken together, both in vitro and in vivo published studies to date, present conflicting findings. Glyphosate alone or GBHs exposure combined may related to adverse developmental or reproductive effects, albeit many studies used very high doses of exposure. In vivo studies have been performed primarily in male rats, from different strains, at different life stages and using different endpoints. It is also not clear whether the possible adverse effects are due to endocrine disruption of GBHs . InterprThe present pilot study examined whether exposure to GBH at a dose of glyphosate considered to be “safe”, i.e. the US Acceptable Daily Intake (ADI) of 1.75 mg/kg bw/day, defined as the chronic Reference Dose (cRfD) determined by the US EPA , affect Active ingredient glyphosate  was supplied from Sigma-Aldrich . The commercial formulation Roundup Bioflow , water (42.5%) and surfactant  was supplied from a local agricultural consortium . The original containers/bottles of glyphosate and Roundup Bioflow were stored in its original container and kept in a ventilated storage cabinet at room temperature (22 °C ± 3 °C) throughout the study. Suppliers provided purity data for each batch of glyphosate and Roundup Bioflow. The opening and the use date of the different batches of test substances were recorded in the raw data. An aliquot of each lot of the test article is maintained in the ventilated storage cabinet, until 5 years from the end of the main experiment. The solutions of glyphosate and Roundup Bioflow were prepared by the addition of appropriate volume of tap drinking water.per each cage. Cages were identified by a card indicating: study protocol code, experimental and pedigree numbers, dosage group. A shallow layer of white fir wood shavings served as bedding . Analysis of chemical characteristics  and possible contamination  of the bedding was performed by CONSULAB Laboratories . Pellet feed and tap drinking water were tested for possible glyphosate contamination as previously described [per each cage) and we did not relocate cages. Noise and handling time were minimized.The entire animal experiment was performed following the rules by the Italian law regulating the use and treatment of animals for scientific purposes . All animal study procedures were performed at the Cesare Maltoni Cancer Research Centre/Ramazzini Institute (CMCRC/RI) . The study protocol was approved by the Ethical Committee of the Ramazzini Institute. The protocol of the experiment was also approved and formally authorized by the ad hoc commission of the Italian Ministry of Health . The CMCRC/RI animal breeding facility was the supplier for the SD rats. Female breeder SD rats were placed individually in polycarbonate cages  with a single unrelated male until evidence of copulation was observed. Each of 24 virgin female SD rats  was mated outbred with one breeder male rat of the same age and strain. Every day, the females were examined for presence of sperm. After evidence of mating, females were housed separately during gestation and delivery. Newborns were housed with their mothers until weaning. Weaned offspring were co-housed, by sex and treatment group, not more than 3 escribed .The cageTwo groups of SD rat dams and relative pups were treated with either glyphosate or Roundup Bioflow diluted in drinking water to achieve the desired glyphosate dose of 1.75 mg/kg bw/day. The F0 female breeders received the treatment through drinking water from gestation day (GD) 6 to the end of lactation, while the offspring (F1) continued to be exposed after weaning for additional 6 or 13 weeks. Glyphosate or Roundup solutions were freshly prepared on a daily basis depending on body weight and water consumption of dams or offspring, measured at scheduled time points. Preparation of drinking water solutions, quantification of glyphosate in water, and dosing adjustments are described in detailed by Panzacchi et al. . During Mean gestational length (duration of pregnancy) was calculated as the number of days from detection of a positive vaginal smear (GD 0) to birth of a litter. Pregnancy was confirmed by the occurrence of parturition. Dams’ body weights were recorded on GD 0, 3, 6 and then daily during gestation until parturition. During lactation, dams’ body weights were recorded at LD 1, 4, 7, 10, 13, 16, 19, 21 and 25 (last measurement before weaning). Pups’ body weight by sex and litter was determined on PND 1, 4, 7, 10, 13, 16, 19, 21 and 25. Dams’ feed and water consumption were recorded twice weekly during gestation , whereas during lactation were measured at LD 1, 4, 7, 10, 13, 16, 19, 21 and 25.To determine the number of pups born to each dam as accurately as possible, we examined cages at frequent intervals during parturition. Dead pups were removed when found and sexed when possible. Sex was determined on PND 1 and sex ratio data was presented as ratio of males to females. The mean litter size was calculated on PND 0 (within 24 h from delivery), 1, 4, 7, 10, 13, 16, 19, 21, 25. Litter size included dead as well as live offspring. Dead pups were visually examined by floating the lungs in saline, to distinguish if they were stillborn (died in utero) or died shortly after birth. Live-birth index was calculated at PND 0 as  × 100. Survival index, calculated as  × 100, was measured on PND 1, 4, 7, 10, 13, 16, 19, 21 and 25. For all the pups, ano-genital distance (AGD), reflecting the linear distance between the genital tubercle and the anus, was measured on PND 4, using a Vernier caliper calibrated with a micrometer stage. Measurement was made from the caudal margin of the anus to the caudal margin of the genital tubercle . Pup bodAfter weaning body weight was measured twice a week, until PND 73 ± 2, then weekly until PND 125 ± 2 and before terminal sacrifices, the means of individual body weights were calculated for each group and sex. Daily water and feed consumption per cage were measured twice a week, until PND 73 ± 2, then weekly until PND 125 ± 2; the means of individual consumptions were calculated for each group and sex. Time to vaginal opening (VO) was determined by daily inspection of all female pups starting on PND 28. The criterion was met for female rats when a complete rupture of the membranous sheath covering the vaginal orifice was observed . The bodStarting on approximately PND 95 and for the duration of 3 weeks, daily vaginal lavage was performed on female rats belonging to the 13-week cohort (10 F/group). To reduce variability, vaginal cytology samples were collected by vaginal lavage at the same time of the day over the course of the experiment, in the mid-morning, between 10:00 and 13:00. Collection, processing and vaginal smear evaluation was performed as described by us previously  .2/O2 (70 and 30% respectively), and sacrificed by drawing blood by cava vein. The time and date of necropsy were recorded. Five days after weaning (corresponding to 49 ± 2 days of treatment), dams were sacrificed and the following organs were collected and alcohol fixed during necropsy: mammary glands , adrenal glands, uterus (including cervix), ovaries, vagina. The adrenal glands, uterus and ovaries were also weighed as soon as possible after dissection. For testosterone concentration determination, blood was collected and serum removed by centrifugation and stored at − 80 °C until analysis.All the animals were anesthetized by inhalation of a mixture of COAll male and female pups belonging to both cohorts were sacrificed on PND 73 ± 2 and PND 125 ± 2. The following organs and tissues were collected and alcohol-fixed: mammary gland , thyroid and parathyroid, adrenal glands, bladder and prostate, seminal vesicle/coagulating gland, left and right testis with epididymis , uterus (including cervix), ovaries and vagina.During necropsy, other tissues displaying anomalies and all gross lesions were collected, if present. Adrenal glands, bladder and prostate, seminal vesicle/coagulating gland, left testis, left epididymis, uterus (including cervix) and ovaries were weighed as soon as collected. In case of paired organs, both organs were preserved. The organ weight was related to body weight and was expressed as both absolute and relative organ weight.Rats were sacrificed randomly across the 4 stages of the estrous cycle. In order to determine and allow correlation with histopathology in reproductive organs and hormone analysis, the stage of estrous cycle was determined by histological appearance of the various components of the reproductive tract for F1 females belonging to the 6-week cohort or by a vaginal smear examined on the day of necropsy for F1 females belonging to the 13-week cohort.Sperm analyses were performed on each male animal from both cohorts, at scheduled necropsies on PND 73 ± 2 and PND 125 ± 2.per animal were recorded, and the numbers of spermatids per gram of testis were calculated. To calculate the daily sperm production (DSP) these values were subsequently divided by 6.1, which is the number of days step 17–19 spermatids are present in the seminiferous epithelium [At necropsy, half of the right testis and the whole right epididymis were frozen in liquid nitrogen and stored at − 80 °C until evaluation. Spermatids resistant to the process of testicular homogenization and spermatozoa present in the caput/corpus and cauda epididymis were counted as previously described by Robb et al.  with sliithelium . Similarithelium .To assess the percentage of morphologically abnormal sperm half of left cauda epididymis of each rat was transferred to a Petri dish containing 2.5 ml  or 3.5 ml  of Dulbecco’s phosphate-buffered saline prewarmed to 37 °C, cut in 2–3 pieces and incubated of approximately 3 min at 37 °C, periodically gently swirling the Petri dish and its contents to facilitate release of sperm from the cauda.per rat were evaluated and scored as morphologically normal or abnormal according to the presence or absence of head or tail defects [Dried smears of epididymal spermatozoa were stained with 1% Eosin Y for 30 min and evaluated at 400 x magnification. Five hundred spermatozoa  defects , 25.After fixation, samples were trimmed, processed, embedded in paraffin wax, sectioned to a thickness of 4–5 μm and then processed in alcohol-xylene series and stained with hematoxylin and eosin for microscopic evaluation. Histopathology evaluation was performed in blind by at least two pathologists. At least one senior pathologist peer reviewed all lesions of oncological interest as well as any lesion of dubious interpretation. In the pathological diagnosis, all the pathologists used the same evaluation criteria and the same classification based on international standard criteria  described in the specific Standard Operating Procedures and long adopted at the CMCRC/RI. The diagnoses are reported in the experimental registries.Serum concentration of free (fT) and total testosterone (TT); 5α-dihydrotestosterone (DHT); 17β-estradiol (E2) and Sex Hormone Binding Globulin (SHBG) were measured in duplicates by solid phase enzyme-linked immunosorbent assays (ELISAs). Blood sera, obtained and stored as described above, were used to assess the quantitative measurements in rat serum of E2, fT, TT, DHT and SHBG by ELISA based on the principle of the competitive binding, using the following commercial kits: “Estradiol rat ELISA” (#DEV9999), manufactured by Demeditec Diagnostics GmbH , “Rat Free Testosterone (F-TESTO) ELISA” (#CSB-E0597r), “Rat Testosterone, T ELISA” (#CSB-E05100R); “Rat dihydrotestosterone (DHT) ELISA” (#CSB-E07879r), and “Rat sex hormone-binding globulin (SHBG) ELISA” (#CSB-E12118r), manufactured by Cusabio Biotech Co. Ltd. .The detection range and the Lower Limit of Detection (LLD) of each ELISA kit was 2.5–1280 pg/mL and 2.5 pg/mL for E2; 0.3–60 pg/mL and 0.15 pg/mL for fT; 0.13–25.6 ng/mL and 0.06 ng/mL for TT; 10–2000 pg/mL and 5 pg/mL for DHT; 375–6000 ng/mL and 375 ng/mL for SHBG. Each kit has been used following the manufacturer’s instructions and absorbance has been measured at 450 nm using a 96-well plate reader .N = 48 total), seven plasma pituitary hormones were measured: adrenocorticotropic hormone (ACTH), brain-derived neurotrophic factor (BDNF), follicle stimulating hormone (FSH), growth hormone (GH), luteinizing hormone (LH), prolactin (PRL) and thyroid stimulating hormone (TSH). FSH and LH were also assessed in 40 pups  randomly selected from the 13-week cohort . Exploratory analyses of circulating BDNF and TSH results from the 6-week cohort showed marginal differences by exposure groups in male pups; thus we attempted to validate these results by measuring BDNF and TSH in all male pups (N = 30) from the 13-week cohort. Plasma TT was measured in duplicates in all dams (N = 24) using an ELISA kit, the “Testosterone Parameter Assay Kit” , following manufacturers’ instructions.Plasma pituitary hormones were measured in duplicates using the “Rat Pituitary Magnetic Bead Panel” , a Luminex® bead-based immunoassay, following manufacturers’ instructions. Using plasma samples from 40 pups  randomly selected from the 6-week cohort . Counting data, not normally distributed, were also analyzed with appropriate regression models. Where the observations were grouped (such as for litter data), fixed and mixed effect models were estimated (litter as random effect) and both reported. For biological parameters related to the body weight (such as the AGD), the statistical analyses were always performed included the body weight of each pup in the regression model. The incidence of pathological lesions, reported as the numbers of animals bearing lesions, were compared using a two-tail Fisher exact test. The statistical analysis was performed using Stata/IC 10.1  and Statisti× 10 ; graphs were obtained using Microsoft Excel and Statisti× 10.p < 0.01) and females (p < 0.01) and in glyphosate-treated males (p < 0.01) (Table p < 0.05) in the Roundup exposed group (Table N = 30), the percent of time spent in each stage of the estrous cycle did not differ between GBH-treated animals and controls  Table . Results1) Table . In femaup Table . The boxls Table  and 4. Tp < 0.05, Tables p = 0.056) in the glyphosate-treated males and a marked and significant increase in Roundup-treated males versus control (p < 0.01). Plasma TSH concentration still showed a borderline significant (p = 0.056) increase in the glyphosate-treated males and a marked and significant increase in Roundup-treated males versus control (p < 0.01).A panel of seven pituitary plasma hormones was assessed in animals from the 6-week cohort . Most pituitary hormones were unaffected by GBH exposure, with the exception of a statistically significant increase in plasma TSH concentration observed in glyphosate-treated males compared with control animals as well as a statistically significant increase in BDNF in Roundup-treated males compared with control animals (p < 0.05); TT showed an increase in the glyphosate-treated group, even if not statistically significant . The box plots and dot plots of DHT was added as supplementary material  compared to control animals  for individual rats was assessed by vaginal smears. We could not statistically evaluate E2  in the different female groups with reference to the stages of the estrous cycle due to insufficient sample size after clustering for endocrine status.BDNF plasma concentration was unaffected in this cohort. Sex steroids were measured in all animals of both 6-week and 13-week cohorts, providing data as follows:p < 0.05) in the Roundup-treated females (13-week cohort), but not 6-week females or in any of the males.The ratio between TT and SHBG (e.g. TT/SHBG), currently used as an indicator of testosterone bioavailability and known as the fT index, was significantly increased (p < 0.05), whereas no effects were observed in E2/SHBG ratio in glyphosate-treated SD male rats.The E2/SHBG ratio, an indicator of E2 bioavailability known as free estradiol index (FEI), significantly increased in Roundup-treated males belonging to the 6-week cohort (p < 0.05) but not in any of the females.The fT/TT ratio significantly decreased in glyphosate- (6-week cohort) and in Roundup-treated males (13-week cohort) (p < 0.01). No statistically significant differences were observed in younger males and females (6-week cohort).Both male and female Roundup-treated animals belonging to the 13-week cohort showed a marked decrease in DHT/TT ratio  and females (Roundup group);age at FE in females was significantly delayed (Roundup group).Roundup Bioflow, when administered to SD rats from in utero through adulthood at a dose level corresponding to the glyphosate RfD defined by the US EPA (1.75 mg/kg bw/day), elicited subtle but potentially adverse effects on reproductive development and hormone concentrations. In particular, two apical endpoints were found to be statistically significantly affected:increased TSH and decreased fT/TT in glyphosate treated rats;increased BDNF and E2/SHBG in Roundup treated ratsStatistically significant changes in hormone profiles, indicators of hormonal activity, were also observed. In the 6 week cohort, glyphosate and Roundup elicited treatment related effects only in males , as follows:decreased DHT; increased TSH; decreased fT/TT in males;increased TT and TT/SHBG in females;decreased DHT/TT ratio in both sexes.In the 13-week cohort, only Roundup and not glyphosate induced sex steroid hormones alterations in both sexes, including:Overall, these effects indicate an impact on pre- and peri-pubertal sexual maturation. Noticeably, the pattern of effects also indicate specific sex-related and treatment-related differences. In particular, the effects of treatment with glyphosate were essentially limited to increased AGD and TSH concentration, and both changes were specific to males. Conversely, Roundup Bioflow seemed to affect both females and males, resulting in a statistically significant increased AGD and sexual hormones imbalances in both the cohorts.Considering these outcomes with a weight of evidence approach, statistically significant differences in apical endpoints (AGD and FE) together with changes in hormonal activity detected in both the treatment groups should be should be taken into account suggesting evidence of concern for reproductive toxicity via an endocrine disruption mechanism . Indeed,In males, a prolonged, albeit of low-intensity, androgenizing effect could eventually evoke a counteracting feedback response from the HPG axis. As apical endpoint, we observed an increased AGD in both the treatment groups. Few animal studies have reported an increased male AGD after chemical exposure. In utero exposure to persistent polychlorinated biphenyls (PCBs) increased AGD in male SD rats , and theFinally, we observed a significant increase in TSH in glyphosate-treated males (6-week cohort) and Roundup-treated males (13-week cohort). Due to resource limitation, we could not investigate T3 and T4 yet, we are planning this work in the near future. We did not observe histological changes in the thyroid gland, therefore the altered concentration of TSH together with a normal histological pattern of the thyroid gland are not indicative of a thyroid-related activity. Nevertheless, our findings prompt further detailed investigation on the effect of GBH on thyroid function and development.BDNF is a neurotrophin playing a fundamental role in survival and differentiation of selected neuronal populations during development, and in the maintenance and plasticity of neuronal networks during adulthood . Our resThe present study has some limitations. First, this is a pilot study performed on a limited number of animals where only one dose was used. However, the dose was selected specifically for its relevance to human health risk assessment, as it is the chronic current RfD defined by the USEPA, 1.75 mg/kg bw/day and therefore a dose level expected to be “safe”. Several hormones were measured in the dams and offspring, but not all hormones were measured in all the animals, due to insufficient material for a complete data set of hormone profiling after the full-scale hematology and clinical biochemistry (data not yet published at the time when this work is presenting). Furthermore, the number and timing of blood sample collection was limited to the final sacrifice of animals, considering that this was a pilot study and that in vivo blood sampling could lead to maternal and pups stress. Another source of uncertainty, which is currently difficult to assess, is the timing of blood sampling during the necropsy session (9.00 am – 3.00 pm); a circadian-dependent modulation of circulating hormones cannot be completely ruled out. Standard errors in different hormone concentrations were wide, in relation to the relatively small group sizes and the physiological variability of hormone concentrations. In females, the estrous cycle status at the time of necropsy is another important source of variability when analyzing sexual hormone profiles. However, even if sacrificing animals on a specific day of the cycle might improve the ability to observe changes in the baseline hormone concentrations, the issue of sacrificing animals in the same cycling period (e.g estrous) is still controversial. The updated OECD Test Guidelines on reproductive-developmental toxicity do not require the sacrifice of females in the same stage of estrous, only the examination of estrous cycle on the day of necropsy is recommend to allow correlation with histopathology in reproductive organs . FinallyThe majority of significant changes observed in hormonal status emerged in the 13-week cohort  compared to animals in the 6-week cohort (sacrificed after puberty) suggesting that more prolonged exposures were more effective in producing imbalances in the hormone concentrations. We have previously reported a possible enhanced retention of GBHs with an increasing pattern of glyphosate excreted in urine in relation to the duration of treatment in these same animals . FinallyThe present study demonstrates that Roundup Bioflow exposure, at a dose level considered as “safe” (1.75 mg/kg bw/day), from prenatal period to adulthood, induced endocrine effects and altered reproductive developmental parameters in male and female SD rats. Roundup Bioflow exposure was associated with androgen-like effects, in particular in females, including a statistically significant increase of AGDs in both males and females, delay of FE and increased testosterone in females. Roundup Bioflow exposure was also associated with altered testosterone metabolism in both males and females, where a statistically significant decrease in DHT/TT ratio was observed in the longest treated group (13-week). Overall, the Roundup Bioflow elicited more and more pronounced effects than the active ingredient itself, which only increased AGD and TSH concentration in male rats in the peripubertal window (6-week cohort). However, considering that retention of any GBH in the body may increase with prolonged exposures, a life-course study on GBHs encompassing intrauterine life through to advanced adulthood is needed to confirm and further explore the initial evidence of endocrine-related effects and developmental alterations emerged in this pilot study.Additional file 1:Figure S1. AGD index and AGD (Mean per litter) box plot (A) and dot plot (B). (DOCX 83 kb)Additional file 2:Figure S2. First estrous box plot (A) and dot plot (B). (DOCX 33 kb)Additional file 3:Figure S3. Male and female DHT box plot (A) and dot blot (B). (DOCX 86 kb)Additional file 4:Figure S4. Effects of glyphosate or Roundup Bioflow exposure on hormones in males (mean ± SEM); coefficient of variation in square brackets. (DOCX 23 kb)Additional file 5:Figure S5. Effects of glyphosate or Roundup Bioflow exposure on hormones in females (mean ± SEM); coefficient of variation in square brackets. (DOCX 23 kb)


Staphylococcus epidermidis at different stages of processing. The protocol was highly efficient at removing bioburden introduced at any stage of processing, with antibiotic treatment and drying the most efficacious steps. The antibacterial activity of non-antibiotic treated AM compared to VDAM was evaluated using minimum inhibitory/biocidal concentrations (MIC/MBC), and disc diffusion assays against Meticillin-resistant Staphylococcus aureus, Meticillin-resistant S. epidermidis, Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis. Antibacterial activity without antibiotic was low, confirmed by high MIC/MBC, and a no inhibition on agar lawns. However, VDAM with antibiotic demonstrated effective antibacterial capacity against all bacteria. Therefore, antibiotic decontamination is a reliable method for sterilisation of AM and the resultant antibiotic reservoir is effective against gram-positive and –negative bacteria.Amniotic membrane (AM) is used to treat a range of ophthalmic indications but must be presented in a non-contaminated state. AM from elective caesarean sections contains natural microbial contamination, requiring removal during processing protocols. The aim of this study was to assess the ability of antibiotic decontamination of AM, during processing by innovative low-temperature vacuum-drying. Bioburden of caesarean section AM was assessed, and found to be present in low levels. Subsequently, the process for producing vacuum-dried AM (VDAM) was assessed for decontamination ability, by artificially loading with  However, the toxic side-effects of antibiotics can be harmful to epithelial cells of the cornea3.Treatment of ocular surface disease, ulcers and infections remains a challenge. Severe bacterial ocular infections are commonly treated with corticosteroids and an intensive course of antibiotics7. Application of AM in the treatment of corneal disease has been reported to reduce infection-causing microorganisms and accelerate healing of infected corneas8, having been shown to be superior to antibiotic treatment alone9.Human amniotic membrane (AM), processed and preserved for therapeutic application is reported to possess anti-inflammatory, anti-scarring, anti-angiogenic and anti-microbial properties, which are valued for their role in wound healing and prevention of post-surgery complications9. Although the mechanism of action of AM is not well explained, it is often attributed to bioactive substances such as growth factors, pro-inflammatory cytokines and anti-inflammatory cytokines, that enhance the function of structural constituents, working as a substrate for local cell migration and adhesion10.AM intervention is an option that mitigates the prolonged use of topical, broad-spectrum antibiotics, preventing steroid-related complications. Studies supporting the use of AM for the treatment of corneal ulcers show significantly enhanced epithelial healing and a reduction in inflammation, corneal scarring, neovascularization, and infection complications11, increased probability of disease transmission, and short shelf-life. Therefore, most clinically used AM is subject to processing and preservation steps prior to use10. Cryopreservation12, heat-drying13 and freeze-drying14 are the most common methods for AM preservation. Although associated with low post-transplantation infection complications15, cryopreservation has been shown to damage the amnion16 which impairs the viability and proliferative capacity of epithelial cells17, decrease levels of many bioactive substances including epidermal growth factors (EGF) and transforming growth factors (TGF), and is constrained by distribution difficulties18. Dried AM followed by radiation sterilisation has been shown to be degraded with reduced growth factor content14. This effect on the stability and function of biological molecules such as growth factors, cytokines and structural proteins, affects the quality of the AM as a biological treatment for eye injury, and justifies the development of an appropriate approach for AM preservation.Freshly collected AM is rarely used for clinical treatment due to post-transplant complications19, AM is often aseptically prepared in order to ensure no contamination is introduced during processing and to limit the spread of any natural bioburden. In vivo during pregnancy, AM is considered sterile, however, potential microbial contamination originates from skin and vaginal flora during delivery of the placenta during birth, with the retrieving staff being an additional source of contamination20. Therefore, any manufacturing process must involve steps to effectively decontaminate such bioburden and allow for long-term preservation of AM.Due to potential degradation by terminal sterilisation techniques21. For dried AM products, vacuum-packing followed by radiation sterilisation is often applied to ensure complete microbial inactivity22.Fresh AM processing generally involves a series of washing steps in physiological solutions to remove raw contamination and blood. Subsequent preservation via heat, freeze-drying, or deep freezing at below −60 °C are applied to suppress water activity to maintain tissue stability, and prevent microbial growth. However, to address bioburden contamination, antibiotics are typically added to the collection medium or storage medium for frozen AMThe sterility and integrity of final manufactured AM is necessary for safe and direct application. However, maintaining AM sterility, quality and functional properties during processing or storage is a challenge. Manufacturing AM in the presence of selected antibiotic/antifungal agents can have several advantageous features, including avoiding deleterious effects on the matrix and biomolecules caused by terminal sterilisation, providing sterility, and inadvertently working as a potential antibiotic reservoir that offers a prophylactic treatment following transplantation.18. This is done by artificially loading AM with high levels of Staphylococcus epidermidis, a skin floral species likely to contaminate amnion23, and investigating the decontamination efficacy of the washing, antibiotic treatment and vacuum-drying steps by counting the surviving colony forming units (CFU) using a spread plate method. The implications of biological and physical antimicrobial activity of fresh AM and the inadvertent antibiotic reservoir effect of vacuum-dried AM (VDAM) against strains of gram-positive and gram-negative bacteria are then studied using minimum inhibitory and biocidal concentrations (MIC/MBC) and disc-diffusion assays.The aim of this study was to validate the microbial decontamination potential of a novel vacuum-drying, aseptic manufacturing technique, and to then investigate further the lasting anti-microbial effects of antibiotics added during processing over long-term storage. We identify the level of microbiological contamination, or bioburden, of elective caesarean section (ECS) AM, and then go on to assess the robustness of decontamination during our optimised, innovative protocol for preserving AM, using vacuum-drying and a broad spectrum antibiotic/antifungal cocktailNeisseria cinerea or Micrococcus spp. Enhanced anaerobic incubation for seven days showed increased growth of Cutibacterium acnes bacteria in samples that were negative at aerobic conditions.Results demonstrating the normal (unloaded) floral contamination of fresh ECS AM can be seen in Table 6 CFU/mL), at different stages of processing, to effectively test the decontamination efficacy of the VDAM process. Figure Due to low and inconsistent levels of natural bioburden on ECS AM, we artificially loaded 5 donated amnion with high bioburden 1006 CFU/mL10 and linear scales). AM samples loaded with S. epidermidis showed significant successive reduction from the original load of 106 CFU/mL as processing progressed. Washing three times in NaCl reduced the bacterial load by almost one log cycle  and the removal of the spongy layer contributed to further lowering the remaining microbial load by 60.53%. Incubating the AM in raffinose solution containing a broad spectrum antibiotic cocktail , P. aeruginosa (ZOI: 2.48 mm) and E. faecalis plates (ZOI: 1.96 mm). All samples of VDAM processed without antibiotic (R) VDAM, processed without raffinose or antibiotic (F), and the PVDF sample discs were not bactericidal to the five strains and did not produce ZOI. However, once the discs were removed there was reduced bacterial growth found below the sample 20. Of those that demonstrated contamination, only Micrococcus spp, Propionibacterium acnes, S. schleiferi and N. cinerea were identified, most likely from skin contact of the placenta during delivery. Previous reports have identified up to 12 species in their surveys including the ones named in our study26. Staphylococcus spp have been identified as the most prevalent contamination of AM26. This study indicates that the risk of major contamination from AM obtained through ECS is low, reducing the risk of contaminated AM being transplanted to the eye. Additional results from PBS samples collected immediately from raw AM before washing (incubated overnight at 35 °C on BA) contained no bioburden (n = 15 from 5 donors), demonstrating that fresh AM could be bacteriostatic before processing.The natural floral contamination of fresh AM obtained by ECS in the Queens’s Medical Centre, Nottingham University Hospitals NHS trust, was shown to be considerably lower than literature reported from normal vaginal deliveriesS. epidermidis were loaded during different stages of processing. S. epidermidis is an ocular pathogen and was selected as a model of staphylococcus genera which is one of the most prevalent causes of bacterial keratitis27. It is considered to be one of the skin floral species most likely to contaminate AM, and has also been shown to adhere well to fresh AM23.VDAM was developed to be stored dry at room temperature in a stable state until use, avoiding the cold chain storage and distribution challenges associated with fresh and frozen AM. A bioburden-free VDAM final product was confirmed during investigation into our protocol, and long-term sterility test results confirm sterility of end-product samples, even when artificially high levels of et al., (2010) who reported that frequent washing can reduce bioburden in processed heart valves28. Most AM preparation protocols use repeated washing in physiological saline to remove blood and immunogens, as well as remains of amniotic fluid. However, we have found that to retain the integrity of the epithelial surface and matrix biomolecules appropriate washing times have to be optimised18. As an added safety precaution for preserved AM, different antibiotic/antimycotic combinations can be used to cover a wide range of gram-positive, gram-negative bacteria and fungi, including penicillin, streptomycin, neomycin, amphotericin B, gentamycin, imipenem, nystatin, polymyxin and vancomycin30. We used a combination of the latter five agents, a cocktail widely used in the UK for tissue allograft banking31. Reports show that antibiotic solutions used for tissue allograft preservation can be stored for up to one-year storage at −20 and −80 without effect on potency. Dried forms of these antibiotics can withstand storage at 37 °C for up to 12 months32.Our results revealed that three successive washes in 0.9% NaCl reduced bioburden by 95.65%, in agreement with Germain 28. Long antibiotic incubation, such as 12 hours at 37 °C, has been shown to be more efficient, but could potentially compromise the cell viability of tissue, including AM34. The current study showed that a two-hour incubation at 37 °C resulted in full eradication of bacteria loaded just before the antibiotic treatment step and that longer incubation times are not required. The antibiotic cocktail was capable of completely eradicating a high load of bacteria.The microbicide activity of antibiotic treatment was found to be more effective at 37 °C than at 4 °C or 22 °CS. epidermidis36. AM can absorb and release antibiotics after rehydration on a wet surface such as agar medium or the corneal surface. Our results showed that the pattern of VDAM release of antibiotic was consistent with the antibiotic control discs, with rapid release at the beginning of application (6 hours) without considerable change in ZOI after 12 and 24 hours of incubation on bacterial lawns. AM has been shown to have slow antibiotic release over seven hours, dependent on the time of AM soaking in antibiotic37, however the release profile may be different for different antibiotics38.AM prepared in antibiotics can serve as a reservoir during transplantation, particularly effective against microbial keratitis frequently caused by different bacteria strains including the conjunctival normal flora 39. AM desiccated to lower or equal to 5% water content prevents microbial growth, thus there is less chance for product contamination. To confirm this, we showed that vacuum-drying alone, without antibiotic treatment, was efficient in removing 94.26% of bacteria, when samples were loaded after the raffinose washing step. Drying in our protocol is primarily applied to AM as a method of preservation and not as a decontamination step, however, so as it was not efficient in reducing all of the bacterial load it would only ever be used as an adjunct to antibiotic treatment for decontamination.Dehydration of AM by air, heat or freeze-drying (lyophilisation) can be applied after washing procedures to decrease the water content of AM, hindering the spread of contamination during storage, and extending the shelf-life. Drying is applied as a final step before packaging, and has been found to maintain AM structural and biological molecules40. No ZOI was observed around washed AM against S. epidermidis38. Tehrani et al. (2013), showed a decrease in antibacterial activity against S. aureus, P. aeruginosa and E. coli of freeze dried AM after 2 hours rehydration, and after 3 times rinsing in PBS41. Our MIC/MBC in vitro study of AM antibacterial activity showed that AM processed without antibiotics has little effect on S. aureus, S. epidermidis, C. diphtheria and Moraxella, indicating that AM has little intrinsic antimicrobial action without the inclusion of antibiotics during the manufacture process. To then investigate the role of the included antibiotics, we then decided to use bacterial strains that may show more resistance to the included antibiotic cocktail in disc diffusion assays. The results of these assays showed antibiotic processed VDAM has a variant ZOI on MRSA (10.74 mm) and MRSE (15.87 mm). Reduced activity was observed on E. coli (8.82 mm), P. aeruginosa (2.48 mm) and E. faecalis (1.96 mm) plates, but was still present and in a consistent manner. AM processed without antibiotic and/or raffinose were ineffective against MRSA, MRSE, E. coli, P. aeruginosa and E. faecalis strains with no ZOI, further indicating that washed AM untreated with antibiotic is not bactericidal.Previous studies have shown that AM antibacterial function after washing or prolonged rehydration was significantly reduced42, and implies that fresh AM may have only a static antimicrobial action by adherence rather than a bactericidal effect, in agreement with Elian et al, 199140. Placing fresh AM on PVDF membrane, to evaluate the biological antibacterial activity by anti-microbial peptide stripping, demonstrated that samples of AM without antibiotics have no such effects against any tested bacteria. These findings disagree with that of N. Kjaergaard et al., 2001, who showed that saline washed AM placed over a nitrocellulose paper was inhibitive to underlying bacterial growth of group B streptococcus, Staphylococcus saprophyticus, P. aeruginosa and S. aureus43.Moreover, visual assessment of confluence of growth underlying the discs showed that bacteria did grow slightly underneath the control discs of plastic, PVDF and untreated AM, but not under VDAM and antibiotic-soaked filter paper. AM antibacterial action by adhesion was higher against gram-positive bacteria compared to gram-negative bacteria. This could be explained by the differences in defence mechanisms between both groups of bacteriaThe current study was limited to investigating whether the antibacterial action of the dried amnion was due to the intrinsic properties of amniotic membrane or due to the antibiotics added during processing. This study could have been further extended by investigating the individual activity of the antibiotics within the cocktail and by considering the effect of long-term storage on each of the individual antibiotics. The study could also be extended by investigating the anti-fungal properties of both amniotic membrane and the antibiotic/antimycotic cocktail added during processing.15. This effect is most likely due to antibiotic use, providing an antibiotic reservoir, substituting for the fresh AM’s lost antibacterial property and effective in prohibiting bacterial growth on wounds44. AM also functions as a physical barrier against infective pathogens45, with elastic, pliable properties allowing it to adhere completely to wounds23, preventing external infection recurrence.AM processed under approved guidelines and as a licensed protocol results in better wound healing results and less post-transplant infectionsThe risk of major bioburden contamination of caesarean section collected AM is low. Nevertheless, the combined AM processing procedure used in this paper is a robust method to remove any bioburden, sub-clinical infection, and manufacturing-related contamination. Non-antibiotic processed AM may be bacteriostatic but has no bactericidal effect. The specific VDAM processing steps of washing in NaCl, spongy layer removal, raffinose incubation and drying, all contribute to significantly reduce the bacterial load, with drying and antibiotic treatment being considered a sterilisation step. The antibiotic reservoir of VDAM can eliminate natural and acquired contamination of gram-negative and gram-positive bacteria, such that VDAM is a reliably sterile product suitable for clinical application. Combined with vacuum packing, VDAM can be confidently and stably stored long-term without the risk of infection-related degradation.46.This study was carried out with the approval of the Nottingham Research Ethics Committee and the study (OY110101) complied with the tenets of the Declaration of Helsinki. Human placental samples were collected from consenting donors undergoing elective caesarean sections. Patients not complying with the inclusion criteria for tissue donation or having a history of antenatal problems were excluded. Written informed consent from the donor was obtained for sample use and the placenta membranes were prepared according to previously published methodologyAM samples (separated from the chorion membrane) from 10 different donors were studied for the natural bacterial flora contamination. Unwashed AM samples were collected in 5 mL PBS  before sonication for 5 minutes. 200 µL of each sample was plated onto blood agar plates  and incubated aerobically for 48 hours and anaerobically for 7 days at 37 °C, before colony forming units (CFU)/mL were counted. 100 µL volumes of sample were inoculated into Anaerobic Basal Broth (Oxoid), for 48 hours and 7 days at 37 °C in aerobic conditions to enhance bacterial proliferation. Bacterial growth was identified by turbidity and API® Staph Rapid 32 A microbial identification kit  was used to identify microorganisms.AM was separated from the chorion membrane by finger blunt dissection and triple washed in 250 mL 0.9% sodium chloride (NaCl) solution  for 15 minutes with rocking agitation to wash out blood. The spongy layer was removed by scalpel blade size 22 attached to a custom-made tool. The AM were soaked in 100 mM raffinose pentahydrate  solution in DPBS containing 5 mL of a broad-spectrum antibiotic/antifungal combination   and incubated overnight at 37 °C. The next day absorbance at 490 nm was measured using a Jenway 6705 UV/Vis Spectrophotometer  and the bacterial suspension adjusted to 108 CFU/mL.A pure culture of a clinical isolate of S. epidermidis was performed individually at three different stages of the VDAM process to assess the efficiency of the decontamination process at different steps . The average of viable colonies was then counted and expressed as CFU/mL.Loaded and fresh AM samples in 1 mL PBS were twice diluted to give 106 CFU/mL of gram-positive; Staphylococcus aureus (S. aureus), S. epidermidis, Corynebacterium diphtheriae, and gram-negative Moraxella lacunata bacterial strains. 50 µL/well of lysogeny broth (LB) medium (Sigma-Aldrich) was added to appropriate wells of a 96-well plate, followed by 50 µL of AM extract at concentrations of 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20, and 0.10 µg/mL loaded in triplicate across the plate. Culture medium only and medium with 100 mg/L gentamicin were used as negative and positive controls respectively. The bacterial strains were grown up in LB medium until optical density = 0.1 at 600 nm. 50 µL/well of the bacterial cell suspension added to each well of the plate. Plates were incubated overnight at 200 rpm on a rocker at 37 °C. Results were assessed visually with the minimum inhibitory concentration (MIC) equivalent to the last clear well, where bacterial growth is completely inhibited, and the minimum bacterial concentration (MBC) equivalent to the lowest concentration of test article required to kill 99.9% of the initial bacterial inoculum.Dry AM discs from 3 donors were prepared with and without antibiotics. Proteins were then extracted from the tissue by grinding with pestle and mortar in a liquid nitrogen bath. AM extracts were then tested against 10S. aureus (MRSA), multidrug resistant S. epidermidis (MRSE), Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa bacteria were prepared from fresh pure cultures in TSB. The viable bacterial count of each was adjusted to 108 CFU/mL using 0.5 McFarland standard. Each of the five bacterial cultures were seeded onto three Mueller-Hinton agar (MHA) (Oxoid) plates in 90 mm Petri dishes using a sterile cotton swab and subsequently the AM discs were aseptically placed on the even lawns of the bacterial strains. An antibiotic-soaked disc of Whatman no.3 filter paper (AB) was performed as a positive control and a disc of transparent laminating plastic (P) was used as a negative control. Three additional discs were also assessed, using hydrophobic highly stripping polyvinylidene fluoride (PVDF) transfer membrane discs, pore size 0.45 µm , one of which was used alone as a control (PV). The PVDF was then used to separate the MHA surface and VDAM-R (R/PV) and VDAM-F (F/PV) to assess biological antibacterial activity, rather than physical blocking. MHA plates were incubated at 35 °C for 18–24 hours and zone of inhibition (ZOI) were measured in mm using a digital calliper . To confirm whether there was any bacterial growth beneath the samples, sterile disposable loops were used to touch areas under the discs of the same bacterial plates and streak on another Tryptone Soya Agar  plate before overnight incubation at 35 °C.The following AM samples were prepared for disc diffusion assays; VDAM processed with raffinose and antibiotic (VDAM), VDAM processed in raffinose without antibiotic (R) and VDAM without raffinose or antibiotic (F). All samples were cut into discs of 10 mm diameter and kept in vials at 4 °C for a maximum of 4 days before the experiment was performed. Cultures of multidrug resistant To specifically investigate time line of antibiotic release from VDAM samples, ZOI were measured at 0, 6, 12 and 24 hours.Triplicate samples of 25 mm diameter  and 10 mm diameter  VDAM discs were tested for sterility. Discs were either aseptically immersed in falcon tubes containing 40 mL TSB for aerobic bacteria, facultative bacteria and fungus, or placed in thioglycolate broth medium U.S.P.  for anaerobic and facultative bacteria. TSB tubes were incubated at 35 °C with shaking (80 rpm), for 21 days along with negative and positive control tubes. Thioglycolate medium tubes were incubated for 21 days at 35 °C without shaking. Turbidity was used as an indicator of non-sterility and was checked daily. Streaking of 10 µL TSB from each tube was performed weekly on TSA plates, which were subsequently incubated for 5 days at 37 °C and observed for bacterial growth.Statistical analysis was performed using GraphPad Prism version 7.01.