Human milk microbiota in sub-acute lactational mastitis induces inflammation and undergoes changes in composition, diversity and load

Sub-acute mastitis (SAM) is a prevalent disease among lactating women, being one of the main reasons for early weaning. Although the etiology and diagnosis of acute mastitis (AM) is well established, little is known about the underlying mechanisms causing SAM. We collected human milk samples from healthy and SAM-suffering mothers, during the course of mastitis and after symptoms disappeared. Total (DNA-based) and active (RNA-based) microbiota were analysed by 16S rRNA gene sequencing and qPCR. Furthermore, mammary epithelial cell lines were exposed to milk pellets, and levels of the pro-inflammatory interleukin IL8 were measured. Bacterial load was significantly higher in the mastitis samples and decreased after clinical symptoms disappeared. Bacterial diversity was lower in SAM milk samples, and differences in bacterial composition and activity were also found. Contrary to AM, the same bacterial species were found in samples from healthy and SAM mothers, although at different proportions, indicating a dysbiotic ecological shift. Finally, mammary epithelial cell exposure to SAM milk pellets showed an over-production of IL8. Our work therefore supports that SAM has a bacterial origin, with increased bacterial loads, reduced diversity and altered composition, which partly recovered after treatment, suggesting a polymicrobial and variable etiology.


Results
Study population. Fifty-one women were enrolled in the study, including 24 healthy-controls, 24 SAM and 3 AM. From the whole data set, some drop-outs occurred during the study (one mother from the control group, and 5 mothers from the SAM group abandoned the study before collecting the second sample). Characteristics of mothers and infants are summarized in Table 1.

Total and active bacterial load increase in human milk during mastitis. Quantification of the 16S
rRNA gene through qPCR of both DNA (total bacterial load) and cDNA (active bacterial load) showed significantly increased bacterial loads in the mastitis samples during the course of the symptoms (Fig. 1). Mean total bacterial load in the control samples was 610,127 cells/ml (SEM = 110,218) and 828,850 cells/ml (SEM = 100,692) at first and second time point, respectively. Mean total load in the mastitis samples (including SAM and AM) during the course of the symptoms reached 3,137,000 cells/ml (SEM = 956,632), which was significantly higher as compared to controls at the same time point (non-parametric Kruskal Wallis test, p < 0.01). After the symptoms had disappeared, mean total load in the mastitis group decreased to 1,430,000 cells/ml (SEM = 259,037), although the values were still significantly higher as compared to controls at time 0, and thus, bacterial load did not fully return to healthy levels at this time point (non-parametric Kruskal-Wallis test, p < 0.01). Mean active bacterial load was significantly lower as compared to total bacterial load in all groups (non-parametric Kruskal-Wallis test, p < 0.001), except in the mastitis group at time 1. Similar values were observed in the control samples at the two studied time points (time 0 = 67,064 cells/ml [SEM = 27,505]; and time 1 = 84,808 cells/ml [SEM = 21,117]). Mean active bacterial load increased in the mastitis group during the course of symptoms, up to 598,395 cells/ml (SEM = 373,253) although this difference was not significant, perhaps as a consequence of the larger data variation, which could be affected by RNA instability. Mean active load in the mastitis group after symptoms disappeared increased up to 1,601,000 cells/ml (SEM = 229,296), and this difference was significant when compared to all the other groups (non-parametric Kruskal-Wallis test, p < 0.001).
Bacterial richness and diversity are reduced during lactational mastitis. After sequencing, one DNA sample (SAM group, time 0) and two cDNA samples (control group and SAM group, time 0) were not considered for further analyses due to the small number of sequences yielded. Total bacterial DNA diversity (as measured by the Shannon Index) and richness (as measured by the Chao1 Index) were lower during the course of mastitis symptoms ( Fig. 2a; non-parametric Kruskal-Wallis test, p < 0.05). Diversity levels did not return to control levels after the symptoms had disappeared, although the estimated bacterial richness significantly increased (non-parametric Kruskal-Wallis test, p < 0.001). A similar pattern was observed at the RNA level: Table 1. Study population's information. *In mothers with mastitis it corresponds to recovery time.  Table S1. At OTU (species) level, the impact of health status on human milk microbiota was also reflected in differences in bacterial composition between healthy controls, mastitis during the course of symptoms (time 0) and mastitis after symptoms cessation (time 1), both at DNA level (Adonis p-value = 0.015, CCA analysis), and active RNA level (Adonis p-value = 0.04, CCA analysis) (Fig. 4). As inferred from DNA analysis, controls appeared more dispersed in the CCA plot, while mastitis groups were more similar in composition and clustered closer to each other. At RNA level, although there was also some overlap, the three groups clustered separately, and the highest divergence was explained by axis 1, which separates controls from mastitis groups. Thus, the CCA plots also support a different bacterial composition at the species level in mastitis and control groups, with a partial recovery after the symptoms disappeared. Streptococcus mitis/oralis, Streptococcus salivarius, Acinetobacter johnsonii, Streptococcus lactarius and Rothia mucilaginosa were the most abundant species detected in the human milk samples at DNA level (Table S2) In addition, LEfSe algorithm was applied in order to further examine potential biomarkers of SAM disease (Fig. 5). Bacteria at significantly higher levels in healthy mother's milk, as compared with mothers suffering  Significant differences in bacterial abundance were also observed when analysing the active bacterial fraction of the samples (Fig. 5b). Under the assumption that pathogens involved in mastitis should be over-represented in the RNA samples relative to their levels in the DNA, an analysis of the bacterial "Activity Index", corresponding to the ratio between the proportion of each bacteria in the RNA-based and DNA-based sequences, was performed. The average activity indexes for the different genera in each group are shown in Fig. 6. The data show that, in acute mastitis, there is an increase in the activity of Staphylococcus, which decreases (negative index values) after the symptoms subsided. Streptococcus, on the other hand, has a negative activity index during acute mastitis, whereas it shifts to positive activity values when the symptoms disappeared. In SAM samples, only a few patterns can be observed, like an increased activity during mastitis and a decreased activity after recovery.      www.nature.com/scientificreports/ In order to further study potential associations between bacterial activity and mastitis, the activity indices of bacterial species in each samples was compared (Fig. 7). Although some milk samples clustered according to health status, results showed a large overlap in bacterial activity ratios between healthy mothers and those with mastitis before and after treatment. This is in agreement with the above-mentioned biomarker discovery analysis in that the etiology of SAM is complex and probably polymicrobial. In addition, the presence of some bacteria at low activity under health conditions that increase in activity during mastitis suggests that some etiological agents of the disease may already be present in health, and that disease onset may be the outcome of a microbial dysbiosis, whose triggering factors should be identified.

Controls
Human milk bacteria exposure to mammary epithelial cells and release of IL8. To study the potential pro-inflammatory effect of bacteria associated to SAM, human milk pellets were co-incubated with www.nature.com/scientificreports/ a mammary epithelial cell line for 24 h. Results showed higher levels of IL8 production in cells exposed to pellets from SAM during the course of the symptoms, which significantly decreased after symptoms disappeared (non-parametric Kruskal-Wallis test, p < 0.01) (Fig. 8). As expected, levels of IL8 from cell supernatants exposed to AM pellets were higher during and after the symptoms, as compared to the other groups, although the small sample size did not allow statistical analyses. Although we did not find any correlation between absolute bacterial loads and the levels of IL8 in the samples, we observed a positive correlation between the genus Staphylococcus' relative abundance and the levels of IL8 in the DNA group (Spearman's ρ, 0.363, p value, 0.003). This positive correlation was also observed at the cDNA level, although it was not statistically significant (Spearman's ρ, 0.229, p value, 0.007).

Discussion
Sub-acute mastitis is a fastidious and common disease among lactating mothers, representing one of the main causes of undesired weaning. Despite its high prevalence and impact on maternal-infant health, SAM is undervalued and under-diagnosed 12 . Studies performed on human milk during lactational mastitis up to date point to an altered bacterial profile, increased abundance in opportunistic pathogens and lower bacterial diversity 14,16,18,20 .
Classically, AM has been associated to Staphylococcus aureus infections 12,[15][16][17] . SAM, on the other hand, has been associated with increased Staphylococcus epidermidis presence [18][19][20] , and with lower abundances of other coagulase negative staphylococci (CNS), and viridans streptococci 21 . Most of these results, however, were derived from culture-dependent analyses, which are known to be biased by false negative rates. Only two studies, up to date, used culture-independent methods to analyse human milk microbial profiles during mastitis 18,20 . In both studies, lower bacterial diversities in mastitis samples were reported, in addition to enrichment in opportunistic pathogens.
In the present study, both total (DNA-based) and active (RNA-based) bacterial composition and load of the SAM and healthy human milk samples have been studied. Results show that total bacterial load was significantly higher during the time of mastitis symptoms, and decreased after the symptoms ceased, although loads remained significantly higher as compared to healthy controls. Interestingly, active load after the symptoms' cessation was significantly higher as compared to healthy controls and mastitis during the symptoms. This could indicate increased bacterial activity levels during the process of re-balancing in the bacterial community. After sequencing the 16S rRNA gene amplicons, we observed lower bacterial diversities both at DNA and RNA level during mastitis, in agreement with previously reported data 18,20 . Richness was also lower during the disease and increased after symptoms had disappeared. However, diversity was not recovered to control levels after the symptoms disappeared. Given that samples were collected short after the symptoms had disappeared (1-4 days after mother's stopped suffering the symptoms was considered as inclusion criteria), data suggest that a full recovery of bacterial communities takes longer than the remission of clinical symptoms and future studies should collect samples at a later stage. Decreased microbial diversity and/or richness associated to microbial dysbiosis have been previously described in several disease conditions, such as inflammatory bowel disease 27,28 , colorectal cancer 29 , tooth decay 30 or celiac disease 31 , among others.
Activity indices, calculated as the ratio between the proportion of each microorganism in the cDNA and the DNA samples from each donor, show differentially active bacteria in the milk from healthy and mastitis-suffering donors. However, milk samples did not cluster according to bacterial activity and maternal health status (controls vs SAM), as observed in the CCA results, and this overlap supports a polymicrobial, variable and dysbiotic etiology of SAM. A plausible explanation for the absence of a unique, clear causal agent responsible of SAM etiology probably lies in the fact that many bacteria associated with SAM are commonly present in human milk under healthy conditions. Thus, contrary to what occurs in AM, SAM may not always be the outcome of an external, or environmental infection but may be caused by a dysbiosis in the pre-existing inhabitants of human milk. Dysbiosis has been introduced as a useful concept to explain disease etiology in cases where classical infection by an external pathogen cannot be identified 26 . In other inflammatory diseases where pathogenic communities are present under health conditions, an environmental factor, ranging from diet changes to immune alterations, Figure 8. Levels if IL8 produced by mammary cells after exposure to bacteria in human milk. The bar plots show levels of IL8 released by cells from a mammary epithelial cell line when exposed to bacterial pellet from healthy controls (Controls_t0, n = 18; Controls_t1, n = 22); sub-acute mastitis (SAM_t0, n = 21; SAM_t1, n = 17); and acute mastitis (AM_t0, n = 2; AM_t1, n = 3). Supernatants from mammary cells exposed only to culture medium were used as negative controls (n = 8). **p < 0.01, non-parametric Kruskal-Wallis test. Created in GraphPad Prism 5 v5.04 (www.graph pad.com).
Despite the high inter-individual bacterial variability of human milk among samples, specific differences between groups were observed. During SAM, Pseudomonas and Acinetobacter were diminished, as compared to healthy controls and after symptoms disappeared (SAM, time 1), respectively. Interestingly, after cessation of the symptoms, SAM (time 1) samples were enriched in typical oral inhabitants, such as Streptococcus and Porphyromonas (DNA); and Prevotella (RNA). In addition, potential anaerobic and opportunistic pathogenic genera like Finegoldia and Peptoniphilus were also increased in SAM at time 1, which could reflect an imbalance in human milk after the mastitis episode. At OTU level, results showed that milk from mothers suffering SAM were enriched in Staphylococcus aureus, even after the symptoms had disappeared, and were also significantly more active as compared to healthy controls. Given that S. aureus was detected in only 1 out of 24 healthy mothers, its higher proportion and prevalence could have been due to an infection from an environmental source. In most other cases, however, the disease-associated bacteria were also found in milk from healthy mothers. Streptococcus lactarius, for instance, was also found to be significantly more active in SAM during and after symptoms cessation, but it was also found at high prevalence in healthy mothers (13 out of 23 samples). In those cases, it is therefore possible that a change in the proportion of pre-existing microorganisms could trigger the inflammatory process. In addition, after symptoms disappeared, samples were enriched in typical oral inhabitants such as Porphyromonas endodontalis and Streptococcus peroris. Given that oral health alterations, especially gingivitis and periodontitis, are very frequent in mothers during pregnancy and lactation 35,36 , the potential role of oral bacteria in triggering inflammation in the mammary tissue should be considered and tested in future studies.
LEfSe biomarker discovery analyses showed several OTUs associated to health, including Acinetobacter johnsonii, Corynebacterium simulans, and Acinetobacter lwoffiii, among others. Corynebacterium kroppenstedtii, S. aureus and oral species such as Prevotella nanceiensis were among the identified SAM biomarkers. Corynebacterium kroppenstedtii has been previously isolated from granulomatous mastitis and breast abscesses samples 37,38 . Others like Acinetobacter johnsonii, Propionibacterium acnes, Lactobacillus helveticus and Lactobacillus zeae were significantly more abundant after symptoms had disappeared. Interestingly, P. acnes is a predominant bacterium in the skin microbiome, which has shown anti-S. aureus activity in vitro 39,40 . Thus, future studies should focus not only on potentially pathogenic organisms, but also on bacteria which could potentially contribute to health conditions, as promoting their growth with pre-or probiotics could prove to be a strategy to prevent or treat SAM 11,41 .
RNA analysis showed that active Lactobacillus iners, Neisseria subflava, Streptococcus lactarius, Streptococcus cristatus and S. aureus were associated with SAM during the symtoms; while others such as P. acnes, Staphylococcus hominis, Acinetobacter lwoffii and Lactobacillus helveticus were associated with health. Some of the potential pathogens deserve further study. Lactobacillus inners, for instance, is a member of the vaginal normal microbiota 42,43 , although it has been associated to vaginal dysbiosis 44 . This species can produce a toxin, named as innerlysin, which may cause cell damage 45 . Thus, its potential role in SAM should be tested in the future. In addition, our results show that pellets from SAM milk samples, containing bacteria, induce pro-inflammatory IL8 release by mammary epithelial cells, supporting an infectious origin of SAM. Although we did not observe an overall correlation between bacterial load and IL8 release in the cells, our results showed a specific correlation between the genus Staphylococcus and IL8 production (only statistically significant at the DNA level), further supporting the potential pro-inflammatory role of this genus.
From a methodological point of view, we propose that the RNA:DNA activity index could be a useful approach to detect pathogenic species in complex human samples, as pathogens would be expected to have higher activity at the disease site than commensal inhabitants.
In summary, our data support a bacterial origin of SAM, with a polymicrobial and variable aetiology, probably the result of a dysbiosis in the milk microbial population.

Materials and methods
Subjects and sampling. A total of 51 mothers participated in the study. Among them, 24 presented symptoms of SAM, 3 presented symptoms of AM and were included for comparison, and the remaining 24 were completely healthy. Human milk samples were collected between 9 and 90 days after delivery, at two time points: during the course of the symptoms (time 0) and after the symptoms cessation (time 1) in the mastitis group; and during a medical consultation to the doctor (time 0), and a second visit a week after (time 1) in the control group. Details of pregnancy and delivery, mother/infant health status, medicines consumption, lactation, and clinical symptoms during mastitis were collected at recruitment through a detailed questionnaire. Mothers (over 18 years old) were recruited at the Breastfeeding Unit of Dr. Peset Hospital and at the Alfafar Health Center (Valencia, Spain). Women were considered to have SAM when presenting breast pain (usually described as profound, needle-like and/or burning pain) accompanied or not by lumps in the breast tissue and without general symptoms 18 . Women were considered to have AM when presenting profound pain in the breast accompanied by at least two of the following symptoms: local inflammation signs (breast redness, local hyperthermia, or sensitive lump), fever and general discomfort 18 . Controls were healthy breastfeeding woman who did not present any of the previous symptoms. Exclusion criteria were: suffering from immunological, metabolic or other severe diseases; or having received antibiotics or probiotics 15 days prior to first sample donation. Breastfeeding counselling was offered to all mothers suffering from breast pain. After any other cause of breast pain (such as an incorrect lactating posture, or infant's short frenulum) was discarded and the diagnosis of sub-acute mastitis was confirmed, mothers were instructed on breastfeeding massage techniques and optimal lactation positioning. Treatments with anti-inflammatory drugs and/or probiotics were prescribed. In fourteen cases from the SAM group, symptoms persisted after 7 days and antibiotics were prescribed. Mothers suffering AM received antibiotics and anti-inflammatory drugs, following the recommended standards. Prior sampling, nipples and Sample processing and DNA/RNA isolation. Human milk samples (4 ml) were centrifuged at 13,000×g for 10 min at 4 °C, discarding fat and whey. Total DNA and RNA were isolated from pellets by using the Master-Pure Complete DNA & RNA Purification Kit (Epicentre, Madison WI, USA) as previously described 46   www.nature.com/scientificreports/ Data analysis and statistics. A quality assessment of the sequences was carried out using the PRINSEQ program 50 . Sequences were end-trimmed in 20 bp sliding windows, and those with average quality value < 30, and length < 250 bp were not considered for further analyses. Reads were pair-end joined using FLASH program applying default parameters 51 . Only overlapping paired-end reads were used for further analysis. The negative controls within the sequencing process produced 615 and 348 reads. The DNA and RNA extraction negative controls yielded 1995 and 1293 reads, respectively. Agrobacterium, Brucellaceae and Bradyrhizobiaceae were identified as contaminants and removed from the analyzed datasets 52 . Operational taxonomic units (OTUs) were generated by clustering reads at 97% of similarity by using VSEARCH 53 . Centroids (representative OTUs) were taxonomically classified at phylum, class, family, genus and species level using feature-classifier command of QIIME2 53 version 2017.8 with Greengenes database (version gg_13_5). Sequences belonging to Streptococcus and Staphylococcus genera, whose 16S gene is highly similar among species, were clustered into OTUs at 100% similarity and > 400 bp alignment length by BLASTn analysis 54 , against a manually curated database for these genera, obtained from RDP Hierarchy Browser 55 . Streptococcus mitis and Streptococcus oralis were identical in the sequenced region and could not be distinguished from each other. α-diversity analysis (Shannon and Chao1 indices), were calculated to estimate sample's diversity and richness; and β-diversity (Bray Curtis dissimilarity index), to quantify the compositional dissimilarity between groups at OTU and genus level, using the R-package vegan 56 .
Canonical correspondence analysis (CCA) was performed by R software vegan package. In order to control the potential effects of maternal antibiotics intake, maternal age and days postpartum, MaAsLin multivariate analysis with linear model 57 was applied. Adonis statistic for permutational multivariate analysis was used to measure differences in variance between groups, and Wilcoxon test implemented in R software was applied to determine significantly different bacterial genera between groups.
Bacterial-OTUs biomarker discovery was performed by linear discriminant analysis effect size (LEfSe) implemented on Galaxy 58 , in order to detect differentially abundant OTUs characterizing the populations of healthy and mastitis-suffering women. Other analyses and graphs were performed in GraphPad Prism 5 v5.04.
In all samples, a bacterial ' Activity Index' was calculated as the ratio between the proportion of each microorganism in the cDNA and the DNA samples from each donor. This ratio would allow the identification of those bacteria whose activity is higher or lower than expected based on their presence, as inferred by sequencing of the 16S rRNA gene. Data were then log-transformed so bacteria which were over-or under-represented in the RNA relative to the DNA fraction of each individual would be assigned positive or negative values of the Activity Index, respectively. Activity Indexes were represented with R in a heatmap, grouped by health status (Controls, SAM and AM) and time (t0, t1), eliminating bacteria at a proportion < 0.1 in more than 90% of the samples.
Exposure of milk bacteria to a mammary gland epithelium cell line. The mammary epithelial cell line MCF7 (ATCC HTB-22), was seeded onto 96-well plates (30,000 viable cells per well) in complete growth medium (DMEM high glucose (Gibco, ThermoScientific) supplemented with 10% v/v inactivated fetal bovine serum (Sigma), 1 mM sodium pyruvate (Gibco), 0.1 mM non-essential amino acids (Gibco), 10 mM HEPES (Gibco), 2 mM l-glutamine (Gibco) and antibiotics (100 U/ml penicillin, 100 g/ml streptomycin (Gibco)). The cells were grown at 37 °C and 5% CO 2 in an incubator for 2 days, and the integrity of the cell culture was checked with an inverted microscope. The medium was replaced with 200 μl fresh complete growth medium without antibiotics, containing the bacterial pellet from 500 μl of centrifuged milk samples. A total of 18 healthy controls, 21 SAM and 2 AM milk samples obtained at time 0; and 22 controls, 17 SAM and 3 AM milk samples obtained at time 1, were analysed in duplicates in the same experiment. Negative controls consisted of MCF7 cells incubated without bacteria. Co-incubation of mammary gland MCF7 epithelial cells with human milk bacteria was maintained for 24 h at 37 °C and 5% CO2 in an incubator. After co-incubation, culture supernatants were aspirated from wells and kept at 4 °C for measuring human IL8 concentration by ELISA (Invitrogen) using 25 uL of supernatants, following the manufacturer's instructions.