The brain is assumed to be a sterile organ in the absence of disease although the impact of immune disruption is uncertain in terms of brain microbial diversity or quantity. To investigate microbial diversity and quantity in the brain, the profile of infectious agents was examined in pathologically normal and abnormal brains from persons with HIV/AIDS [HIV] (n = 12), other disease controls [ODC] (n = 14) and in cerebral surgical resections for epilepsy [SURG] (n = 6). Deep sequencing of cerebral white matter-derived RNA from the HIV (n = 4) and ODC (n = 4) patients and SURG (n = 2) groups revealed bacterially-encoded 16 s RNA sequences in all brain specimens with α-proteobacteria representing over 70% of bacterial sequences while the other 30% of bacterial classes varied widely. Bacterial rRNA was detected in white matter glial cells by in situ hybridization and peptidoglycan immunoreactivity was also localized principally in glia in human brains. Analyses of amplified bacterial 16 s rRNA sequences disclosed that Proteobacteria was the principal bacterial phylum in all human brain samples with similar bacterial rRNA quantities in HIV and ODC groups despite increased host neuroimmune responses in the HIV group. Exogenous viruses including bacteriophage and human herpes viruses-4, -5 and -6 were detected variably in autopsied brains from both clinical groups. Brains from SIV- and SHIV-infected macaques displayed a profile of bacterial phyla also dominated by Proteobacteria but bacterial sequences were not detected in experimentally FIV-infected cat or RAG1 −/− mouse brains. Intracerebral implantation of human brain homogenates into RAG1 −/− mice revealed a preponderance of α-proteobacteria 16 s RNA sequences in the brains of recipient mice at 7 weeks post-implantation, which was abrogated by prior heat-treatment of the brain homogenate. Thus, α-proteobacteria represented the major bacterial component of the primate brain’s microbiome regardless of underlying immune status, which could be transferred into naïve hosts leading to microbial persistence in the brain.

Funding: These studies were supported by the Canadian Institutes of Health Research (G118160525). CP holds a Canada Research Chair (CRC) (Tier 1) in Neurological Infection and Immunity and is an Alberta Innovates-Health Solutions Senior Scholar. MS holds a Canada Research Chair CRC (Tier 1) in Interdisciplinary Microbiome Research. None of the authors have commercial interests or activities related to the contents of the present manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Human immunodeficiency virus type 1 (HIV-1) infection with the ensuing development of the acquired immunodeficiency syndrome (AIDS) is a recognized cause of perturbation within microbiomes within the gut and other organs [17] . The altered immune status of HIV/AIDS patients is associated with qualitative and quantitative shifts in microbiome diversity and quantity as well as increasing the likelihood of microbial translocation from the gut to other organs [18] . These events occur because of reduced immune surveillance and damaged barrier function at epithelial and endothelial sites, including damage to the blood-brain barrier [19] . Multiple studies have shown the blood-brain barrier to be more permeable in HIV/AIDS, implying this circumstance might contribute to HIV-associated neurological disease [20] , [21] . Given the emerging appreciation of microbiome disruption together with the damage to the brain injury during HIV/AIDS, we hypothesized that brain tissue from patients with HIV/AIDS might contain greater microbial quantities and perhaps diversity, derived largely from other organs such as the gut, which has a well-established microbiome and altered function during HIV infection. In fact, the present studies showed that all examined human brains contained bacterially-expressed RNA and associated products together with a predominance of α-proteobacteria, regardless of the patients’ immune status and concurrent diseases. Moreover, a similar bacterial population was observed in the brains of non-human primates. This microbial population found in the human brain could be transmitted into immunodeficient mice and subsequently recovered 7 weeks later suggesting the brain was tolerant to the presence of certain bacterial species

The existence of commensal microbes that colonize organs within the human body has long been recognized and termed the microbiome [1] , [2] . Once thought of as harmless tenants, it has become increasingly obvious that the microbiome is not comprised of passive passengers but of specific infectious agents that participate in shaping the metabolic and immune status of the host [3] , [4] . The composition of the human microbiome at several body sites including skin, oral cavity, gastrointestinal tract, nasopharyngeal passage and urogenital tract are subjects of intensive study [2] , [5] , [6] , [7] , revealing both local effects at these sites but also systemic effects, including influencing inflammatory or metabolic functions in privileged organs such as the brain [8] , [9] , [10] . These latter studies have reported complex bacterial populations with wide variations in individual bacterial constituents at the species level, both in serially sampled humans and across groups of humans. The composition of the microbiome at the phylum level is consistent throughout the body with the exception of the skin, where diverse population structures occur at different sites [11] , [12] . In most body sites sampled to date, Firmicutes and Bacteriodetes represent the dominant phyla with substantial numbers of Actinobacteria and Proteobacteria detected, while other phyla comprise a small proportion of the remaining species. Importantly, body sites previously presumed to be sterile in healthy humans, such as the vascular endothelium, have been shown to be colonized without apparent signs of disease. In contrast, diseases such as biliary cirrhosis [13] , [14] , atherosclerosis [15] and aortic aneurysms [16] , previously considered attributable to autoimmunity or pathogenic metabolites, are now hypothesized to be induced or exacerbated by infectious agents, possibly as part of local microbiomes

(A) Comparison of the relative quantity of 16 s rRNA in human and matched recipient mouse brains showed that mean bacterial rRNA levels in recipient mice were present at ∼40% of the mean ODC14 brain homogenate rRNA levels but less than 1% for the heated-treated ODC13 brain homogenate (prior to heat-treatment), measured by real time RT-PCR. (B) Mouse host inflammatory gene transcripts (ifn-α, il-1β and il-12) in mouse brains were not induced by implantation of untreated compared to heat-treated human brain homogenates. (C) The bacterial 16 s rRNA sequences from the ODC14 brain homogenates and the recipient mouse ODC14 brains were similar with substantial phylogenetic overlap between the bacterial genera, as evidenced by the 97% similarity for Delftia acidovorans sequences.

As bacteria were evident in brains of persons with and without apparent neurological disease ( Figure 1A ) and bacterial sequences were not present in RAG1 −/− mice ( Figure 4B ), we investigated the transmissibility of brain-derived bacteria in these animals because of their apparent sterile brains and their minimal adaptive immune responses, which might potentiate any disease mechanisms. Homogenized human brain samples (ODC13 and 14) with and without prior heat treatment, respectively, of the homogenate were stereotactically implanted into the striatum of RAG1 −/− mice. The implanted mice were maintained for seven weeks and then sacrificed and total RNA was extracted from the brains. In mice that received unheated (ODC14) brain homogenates, 16 s RNA V3–V4 sequences were detected at levels that were at 40% abundance of the original implanted human brain homogenate ( Figure 6A ). Conversely, 2 of the three mice that received heat-treated brain homogenates (ODC13 H.T. ) did not contain detectable 16 s rRNA while the third animal contained 16 s rRNA at levels >1% of the original implanted human (ODC13) tissue. The expression of ifn-α, il-1β and il-12 (mouse) transcripts was examined in the brains of the both groups of recipient mice; these analyses revealed implantation of untreated brain homogenates did not evoke increased or sustained neuroimmune responses compared to animals’ brains that received the heat-treated brain homogenates ( Figure 6B ). Expression of essential host gene transcripts such as calreticulin was also similar in both groups and neurological deficits were not evident in either experimental group of mice. Sequencing of 16 s rRNA from the initial untreated human brain homogenate (ODC14) and the corresponding recipient mouse brains showed closely related sequences with phylogenic clustering, again pointing to a high proportion of Proteobacteria-derived sequences ( Figure 6C ). Taken together, these results indicated that human brain-associated bacteria could be transferred into and recovered from the brains of recipient mice at comparable bacterial RNA quantities and diversity to the original implanted human brain homogenate.

(A) Ethidium bromide-stained agarose gel showed the amplicon generated by primers 16 s514F and 16 s 806R from SIV-infected macaques 1–10, SHIV-infected macaques 1–3 and FIV-challenged macaque 1 and 2. cDNA synthesized with ultrapure water, water carried through both rounds of nested PCR and water used as template only in the final round of PCR were all included as negative controls. (B) Phylogenetic analysis of 16 s rRNA region sequences amplified by the primers 514F and 806R derived from macaque brain specimens. Clustal alignments were generated comparing amplicon sequences with the equivalent position of published 16 s rRNA sequences identified by BLAST analysis. The Neighbor joining tree was generated based on 10,000 bootstrap trials and rooted on the macaque mitochondrial 12 s rRNA sequence. Again, these data showed a predominance of Proteobacteria in brain-derived 16 s rRNA clones although other bacteria, e.g., Actinobacter and Bacilli, were detected.

Since all of the human brain specimens examined herein exhibited bacterial sequences while non-primate experimental animals showed no evidence of bacterial infection despite being immunosuppressed and brains were collected with similar protocols for handling and preparing tissues, bacterial RNA sequences were investigated among experimental non-human primates. In all but one of the cynomolgus macaques’ brains infected with SIV (n = 10) and all animals infected with SHIV (n = 3) 16 s rRNA V3–V4 sequences were observed, ( Figure 5A ) but of the FIV-challenged macaques (n = 2), the brain of one animal (Control 2) did not exhibit detectable bacterial sequences; this latter animal was housed in a SPF facility. Phylogenetic analysis of 16 s rRNA sequences derived from macaques showed more diversity than among the human brains but again displayed sequences largely matching Proteobacteria ( Figure 5B ). These studies of non-human primates recapitulated the above observations in human brains derived from both deep sequencing and amplification followed by cloning and conventional sequencing.

Based on amplification and cloning of at least 5 clones per patient, a neighbor-joining phylogenetic tree was constructed predicated on representative clones exhibiting consensus sequences as well as unique clones with the nearest bacterial 16 s rRNA matches identified by BLAST analysis, which was rooted to human mitochondrial 16 s rRNA ( Figure 4C ). For comparison, the same analysis was conducted using cDNA derived from serum and peripheral blood mononuclear cells (PBMCs) from healthy donors (n = 2). 16 s rRNA V3–V4 sequences from HIV and ODC patients overlapped phylogenetically and with the cloned sequences derived from SURG specimens. Most sequences derived from blood corresponded to the Bacilli class and diverged from brain-derived sequences ( Figure 4C ). Proteobacteria sequences predominated among the brain-derived clones. When interpreted at the class level, the predominant class was α-proteobacteria, with some γ- and δ-proteobacteria representation. Of note, the sequences amplified from ODC patient 1 (ODC1) were the only sequences belonging to the Bacteriodetes phylum. The patient’s history of hemorrhagic cerebral infarction, together with the distinct profile of 16 s RNA sequences observed in this patient might represent disruption of the blood-brain barrier, resulting in infiltration of atypical organisms that differed from the other brain specimens. Amplification of the 16 s rRNA V8 sequences again showed similar amplicon levels among ODC and HIV brain specimens ( Figure S4A ). Amplified V8 sequences from brain were identified as belonging to different classes of bacteria belonging to α-, β-, γ- and δ-proteobacteria, and Bacilli although β-protobacteria was the predominant class ( Figure S4B ). Thus, these studies validated the identities of the bacterial classes detected in human brains by deep sequencing.

(A) Representative ethidium bromide gels show the amplification of a single band by a nested PCR protocol but in matched (nested) water controls, a product was not observed. (B) Real time RT-PCR showed similar levels of mean 16 s rRNA amplicon quantities in HIV and ODC brain samples, however SURG samples showed lower amplicon levels of 16 s rRNA products. (C) Phylogenetic analyses were determined among cloned amplicons generated from brain cDNA from ODC (n = 6), HIV (n = 6), SURG (n = 3) as well as cDNA from PBMCs (n = 2) and serum (n = 2) from healthy volunteers (PBMC 1,2; Serum 1) using the universal bacterial 16 s primers 514F and 806R and the equivalent region of various published 16 s rRNA genes. The alignments were used to generate a neighbour joining tree using 10,000 bootstrap trials. There was a predominance of α-proteobacteria in brain-derived 16 s rRNA clones although other classes, e.g., β- and γ-proteobacteria, and Bacilli, were amplified from blood-derived cDNA.

To confirm the above deep sequencing detection of bacterial RNA, real time RT-PCR was performed amplifying distinct regions of bacterial 16 s ribosomal RNA (rRNA) in cDNA generated from autopsy-derived cerebral white matter of HIV (n = 6) and ODC (n = 6) patients, as well as cerebral white matter from surgical specimens (SURG (n = 6)). PCR conditions using several primer pairs were optimized in advance by colony PCRs using E. coli DH5α as a template. All PCR reagents were confirmed to be free of contaminants using ultrapure water as no-template controls for all primer pairs ( Figure S3A ). In all brain samples examined, bacterial 16 s rRNA encoding sequences were detected, although PCR products were not detected in reaction in which there was no template ( Figure 4A ). The mean level of 16 s rRNA-encoding amplicons was similar in the HIV and ODC autopsied brain samples ( Figure 4B ) but greater than in the SURG specimens; 16 s rRNA sequences were not detected in the brains of FIV-infected cats (n = 3) and RAG1 −/− mice (n = 3) ( Figure 4B ). Of note, amplification of bacterially-encoded groEL transcripts generated amplicons that were consistently detected at high cycle thresholds of ≥34–35, suggesting that bacterial transcript levels were low in human brains. To control for contaminated reagents and equipment used throughout the tissue collection and RT-PCR process cDNA synthesis was performed using ultrapure water and ultrapure water used to rinse a surgical tool (hemostat) opened in the operating theatre during the collection of SURG6 as the substrate in parallel with a subset of brain-derived RNA using the same reagents and equipment. This control product, used as a PCR template, failed to amplify an amplicon corresponding to 16 s rRNA or any other amplicons ( Figure S3B ). These studies verified the presence of bacterial RNA in human brain samples but also validated the methodologies used herein by showing all of the negative controls did not yield PCR products.

In situ hybridization (ISH) performed on brain sections, using a probe hybridizing to a conserved sequence within the bacterial 16 s rRNA encoding gene, also revealed positive detection of spherical structures, 2–6 µm in size, resembling bacteria and clusters thereof in ODC ( Figure 3E , arrowhead) and HIV ( Figure 3G arrowhead) patients’ brains but again fewer and smaller than GFAP-immunopositive fibrous astrocytes in both clinical groups ( Figure 3F and H ). As observed above with peptidoglycan staining, ISH-positive structures were found within cells and in the extracellular matrix at high magnification ( Figure 3J , arrowhead). Sections from the same patients hybridized with a probe encoding a scrambled sequence did not show specific labeling ( Figure 3M (ODC) and 3N (HIV)).

Autopsy-derived ODC (A), and HIV (C) brain specimens were immunolabeled with anti-peptidoglycan antibody. Peptidoglycan (PGN)-positive bodies (arrows) were morphologically consistent with bacteria and smaller than CD45 immunopositive microglia from ODC (B) and HIV (D) patients, imaged at the same magnification. Double DIG-labeled EUB 338 probe in situ hybridization (ISH) against the 16 s rRNA gene was hybridized with slides from the same ODC (E) and HIV (G) patients and labeled with alkaline phosphatase-conjugated sheep anti-DIG FAB` fragments and stained with NBT/BCIP. ISH-positive bodies featured morphology resembling bacteria (arrow heads) and were smaller than GFAP immunopositive astrocytes in ODC (F) and HIV (H) patient sections. Peptidoglycan-labeled cells with both spherical and rod morphology were observed within the brain parenchyma and in a blood vessel (I) (White arrow) of an ODC patient. Peptidoglycan immunopositive bodies were observed within the cytoplasm of GFAP-immunolabeled astrocytes (I, inset) and Iba-1 immunolabeled microglia (J, inset). Spherical clusters of EUB 338 hybridized cells J) were evident (black arrowheads). Slides from the same ODC11 (K) and HIV11 (L) patients were processed under identical conditions except that the primary antibody was omitted. A scrambled DIG-labeled probe was hybridized to slides from the ODC11 (M) and HIV11 (N) under identical conditions used for the EUB 338 probe. In all cases specific signals were not detected. (Original magnification 200×). (O) A section from the forebrain of one of the FIV-infected cats was immunostained with the anti-PGN antibody and developed with DAB with no detectable signal. (Bar: A–H, 50 microns; I, 25 microns; J, 20 microns; K–N, 50 microns) (Magnification: A–H, 200×; I, 400×; J, 600×; I and J insets, 600×).

The above molecular findings prompted investigation of bacterial antigens and genetic material within the cerebral white matter of HIV and ODC persons. Immunoreactivity to the ubiquitous bacterial wall constituent, peptidoglycan (PGN), was observed in white matter of both ODC ( Figure 3A , arrow) and HIV ( Figure 3C , arrow) patients as particulate immunostaining, albeit less abundant than CD45-immunopostive microglia in both groups ( Figure 3B and D ; arrowhead indicating microglial cell body). Peptidoglycan immunoreactivity was also evident within blood vessels ( Figure 3I , arrow) and was co-localized with immunoreactivity for GFAP (astrocytes) ( Figure 3I , inset) and Iba-1 (microglia and macrophages) ( Figure 3J , inset). Other HIV patients with or without encephalitis showed similar patterns of peptidoglycan staining to that observed among ODC patients ( Figure S2 ). Control slides in which the primary antibody was omitted did not show immunoreactivity ( Figure 3K [ODC] and 3L [HIV]), nor did cerebral sections from FIV-infected cats reared in a specific pathogen free facility ( Figure 3O ).

As both bacterial and viral genomes were detected in the present brain specimens, we examined host genes known to participate in responses to infections. Several genes exhibited variable expression in HIV relative to ODC brains based on tag counts from the whole transcriptome analysis with myd88 showing marked suppression (4-fold) in HIV-derived brain specimens compared to the ODC group; conversely, lysozyme was induced in the HIV group ( Figure 2A ). Analysis of host genes germane to viral infections disclosed that antiviral genes such as mx1 and oas1 were induced in the HIV brains while MHC Class I genes (hla-c, -b and -a) were relatively suppressed in the same group ( Figure 2B ). Further analysis by real time RT-PCR showed that transcripts encoded by multiple genes implicated in host responses to viral infections of the brain were increased in the HIV/AIDS brains (HIV4, 7–10, n = 5)) with cd3ε, egf and il-23 showing significant increases compared to ODC brains (ODC6-10, n = 5)) ( Figure 2C ). Network analyses showed associations between bacterial sequence tag quantities and variable expression of host genes implicated in essential cellular structure and maintenance functions ( Figure S1 ). From these studies, it was evident that there was a differential expression of host neuroimmune genes in the clinical groups, emphasizing the divergent biological environments in the HIV compared to the ODC brain specimens.

( A ) Total sequence tags that were unambiguously identified as belonging to a bacterial phylum were grouped for each patient from which the percentages for each phylum were displayed. All patients showed a predominance of Proteobacteria-associated sequences. (B) Despite inter-individual variability the mean percentage of Proteobacteria sequences among the HIV, ODC and SURG groups was similar. (C) The majority of bacterial sequences identified in all patient samples belonged to the Proteobacteria phylum, which showed the greatest similarity to the α-proteobacteria class. (D) The majority of bacteriophage sequences identified matched Proteobacteria-tropic phage sequences although bacteriophage sequences were not detected in the SURG samples.

The relative abundance of several bacterial phyla in the present brain samples was determined using normalized tag counts from each sample ( Figure 1 ). The most abundantly detected phylum in all samples was Proteobacteria, based on a patient-by-patient analysis ( Figure 1A ). Moreover, one of the dominant phyla in other organs (Firmicutes) was undetectable in most brain-derived RNA samples by this approach. The next most abundant phylum in most samples was Actinobacteria. The identities of minor bacterial constituents showed variation between different patients. Surgically-derived cerebral samples (SURG-1 and SURG-2) showed a similar predominance of Proteobacteria, but in contrast to the autopsy-derived specimens, Actinobacteria were not among the principal constituents ( Figure 1B ). The sequences mapping to members of the Proteobacteria phylum were examined at the class level, revealing that α-proteobacteria was the most frequently detected class of Proteobacteria in all brain samples ( Figure 1C ). In agreement with the distribution of bacteria in the present samples, the majority of bacteriophage sequences detected in autopsy-derived cerebral white matter specimens corresponded to Proteobacteria-tropic phage RNA ( Figure 1D ). Tags mapping to bacteriophage sequences were not identified in the surgically-derived samples. HIV-1 sequence-specific tags were also detected in brain specimens from the HIV group but other viral RNA sequences were infrequently detected by deep sequencing in the present specimens with the exception of the specimen from the ODC patient (ODC4) diagnosed with rabies encephalitis, from which in excess of 20,000 rabies virus sequence tags, spanning the entire viral genome, were identified [23] . To explore abundance of viruses known to infect the brain in more detail, the presence of viral genomes was investigated in total RNA and genomic DNA from HIV and ODC white matter samples. Cytomegalovirus (HHV-5), HHV-6A/B and Epstein-Barr virus (HHV-4) sequences were variably present in specimens from both groups of autopsied samples but other DNA and RNA viruses were infrequently detected in HIV or ODC brain specimens ( Table 3 ).

Analysis of the present dataset revealed sequence tags mapping unambiguously to 173 different bacterial- and phage- -derived sequences. Several bacterial classes were represented as high tag numbers in multiple patient samples compared to the host gene, βIII-tubulin, which was expressed in all samples ( Table 2 ). α-proteobacteria sequence tags were identified in all patient samples and showed the greatest similarity to members of the Sphingomonadaceae family, which were of particular interest because of the ability of several members of this family to synthesize and incorporate sphingolipids including ceramide into their outer membranes [22] . The most abundant bacterial tags matched most closely with the Sphingomonas wittichii RW1 genome and the majority of which were found to map to sequences within the 23 s rRNA loci. However, the matches to bacterial sequences had restricted discriminating power because they were based on short sequence tags and were considered to be matches limited only to the class level.

Massively parallel sequencing without prior amplification was performed using cDNA derived from total RNA that was extracted from autopsy-derived cerebral white matter of HIV/AIDS (HIV) (n = 4: HIV1-4) and other disease control (ODC) (n = 4: ODC1-4) patients, as well as from white matter extracted from brain tissue collected during surgical (SURG) resections for epilepsy (n = 2: SURG 1–2) ( Table 1 ). The sequence tag lengths varied between 36 and 77 nucleotides and the maximum number of unambiguously identified tags ranged from 5,500,000 to 11,700,000 depending on the individual patient sample. Of these 3,000,000–7,000,000 tags per patient mapped to the human genome (∼20,000 open reading frames); 200 to 21,000 of the remaining tags mapped unambiguously to bacterial or viral sequences. Following contaminant screening based on non-human libraries, all sequence tags were shown to be intrinsic to the brain-derived libraries.

Discussion

The present study reports the first deep sequencing analysis of microbial populations within the normal appearing tissue in the human brain with confirmatory methodologies that highlighted the consistent presence of bacterial ribosomal RNA and associated bacterial products. The majority of the bacterial RNA sequences identified in all human and nonhuman primate brains were encoded by members of the α-proteobacteria class, regardless of the underlying disease process. This restricted bacterial diversity observed in normal brain tissues is in contrast to the findings of a wide variety of organisms from other groups studying brain abscesses [24], [25]; many of which display colonization by bacteria from other tissues. This dichotomy in findings suggests that the bacteria identified in normal appearing tissue in the present studies might be are outcompeted and/or supplanted by organisms from other body sites in the case of intracerebral abscesses. Peptidoglycan immunoreactivity and in situ hybridization detection of bacterial rRNA were apparent within glial cells and in the extracellular space. In vivo cerebral implantation of human brain homogenates into mice showed that the 16 s RNA sequences recovered from recipient animals’ brains were conserved and expressed at levels similar to the initial human brain homogenate. Transmission was interrupted by heat treatment of the initial homogenate, implying that viable bacteria were required for transmission. These findings indicated that bacteria were present in the primate brain and do not appear to be derived from the predominant populations at other human body sites. Indeed, the predominance of α-proteobacteria in the brain is unparalleled in other body sites where the microbiomes are dominated by Firmicutes, Bacteriodetes and Actinobacteria.

The bacterial rRNA V3–V4 region-based detection of α-proteobacteria in the present studies was supported by the unbiased (non-amplified) deep sequencing. The relatively greater abundance of β-proteobacteria identified using the V8 region amplicon might reflect a bias because of the poor discriminating power of the smaller V8 amplicon or the restricted diversity within this region relative to the analyzed V3–V4 region amplicon, leading to imprecise assignment of sequences to this closely related class. There might also be a bias introduced by the PCR conditions, despite the use of universal primers, resulting in an over-representation of β-proteobacteria sequences. This finding needs to be clarified through culturing of brain specimens as part of future analyses to identify definitively the components of the primate brain’s microbiome at the species level. Of interest, α-proteobacteria comprise one of the most diverse bacterial classes with wide spread biological niches and actions including detoxifying effects in the environment [22], [26]. The acquisition of this class of infectious agents by the brain might represent a beneficial organ-specific adaptation.

The potential for contamination of samples at any stage of the tissue preparation, together with blood contamination was considered throughout the present studies. Controls for all lots of reagents were used throughout this work at all steps of the RNA extraction, cDNA synthesis and conventional and quantitative RT-PCR. The current tissues varied in harvest times and sources; for example, autopsy times ranged from 12–24 hr, while surgical samples were collected under sterile conditions and immediately frozen on dry ice in the operating room. To confirm that 16 s rRNA amplicons identified were intrinsic to the tissue and not introduced during sample preparation RNA extraction, cDNA synthesis and PCR, extensive reagent and equipment controls were used throughout these studies up to and including having the entire process repeated by different personnel in another facility using different lots of consumables and reagents (Figure S3B). White matter was intentionally selected to limit blood contamination of samples; additionally, bacterial genome and products were detected in brain parenchyma remote from blood vessels and within cells (glia) known to phagocytose foreign materials. Likewise, the current simian brain samples were harvested and processed by different investigators in a distant facility, yet the same phyla were predominant in the majority of macaque brains examined. Brain samples from experimental animals reared in SPF conditions with concurrent immunosuppression (RAG1−/− mice and FIV-infected cats) were included as controls within these studies but did not show bacterial 16 s rRNA sequences in brain despite being processed in an identical manner to the human and macaque brains, even with prior amplification steps and identical preparatory methods. Despite the diversity of techniques and sources of the present brain tissues, α-proteobacteria represented the most prevalent bacterial class discovered within the human brain, which was in contrast to blood samples. The consistency of α-proteobacteria detection in primate brains but not in SPF experimental animal brains emphasized the reliability and specificity of the present observations.

Several of the bacterial classes observed in the present studies have been associated previously with human diseases. For example, an organism similar to many of the 16 s V8 region sequences, Delftia acidovorans has been implicated in endocarditis [27], bacteremia [28], [29], corneal keratitis [30] and urinary tract infections [31]. In addition to causing infections in compromised patients, D. acidovorans and other members of the Comamonadaceae have been identified as part of the bacterial community in the arterial wall in patients who have had aortic aneurysms [16]. D. acidovorans has also been isolated from cerebrospinal fluid, sputum, urine, pharynx and wounds without concurrent signs of disease [32]. Taken together, this ubiquitous environmental organism likely represents a commensal organism with wide tissue distribution that can act as an opportunistic pathogen in vulnerable patients. As such, the prevalence of sequences showing similarity to Delftia sp. in the majority of patients in all the present clinical groups bears close scrutiny as host specific factors might determine whether or not this organism contributes to brain disease. Two HIV/AIDS patients exhibited sequences similar to Alcaligenaceae; members of this family have been identified as components of the normal flora in Peyer’s patches [33], but members have also been implicated in endocarditis [34], bacteremia [35], [36] and meningitis with or without ventriculitis in neonates [37], [38], HIV-infected persons [39], other immunocompromised adults [40] or following invasive surgery [41]. Thus, identifying members of this family may represent translocation of commensal gut organisms to the brain or a previously unidentified subclinical infection in these patients.

As the surgical cerebral resections were collected from patients as part of a procedure for epilepsy, they represent the samples with the lowest probability of ex vivo changes, such as RNA degradation or a possible artifact of bacterial growth. Differences in the bacterial populations between the surgically- and autopsy-derived specimens could represent post mortem–related differential levels of RNA degradation or changes in microbial replication. The >5 fold increase in the representation of Archea in the cerebral surgical specimens relative to the ODC group was interesting, as members of this kingdom were undetectable in all but one autopsy brain specimen.

In an organ widely assumed to be free of infectious agents in the absence of a specific disease process, autopsied and surgically-derived human brain specimens showed a restricted but distinct bacterial population in the present studies, which was composed of bacterial classes chiefly recognized in the physical environment, i.e., soil and water. The sources of these agents might include oral consumption or inhalation with eventual transport to the brain as intracellular agents in activated leukocytes trafficking into the brain. The brain is constantly surveyed by trafficking leukocytes (activated lymphocytes and macrophages), which provide a Trojan horse mechanism for microbial entry into the nervous system across the blood brain barrier. In fact, this mechanism is well recognized as a route by which viruses infect the brain and likely underlies the detection of herpes viruses in both the HIV and ODC brains. Corroborating this latter point is the report of peptidoglycan detection in brain lesions from multiple sclerosis patients, which are heavily infiltrated with blood-derived leukocytes [42]. Since bacteria express multiple molecules that activate immune signaling cascades by engaging Toll- or NOD-like receptors, etc., their capacity for influencing brain function [43] is immense. Hence, studies focused on delineating the brain’s microbiome at the species level together with their individual effects on host cell physiology might lead to a greater understanding of human neurobiology including cognitive, motor, sensory and behavioral functions.