Infection of Fungi and Bacteria in Brain Tissue From Elderly Persons and Patients With Alzheimer's Disease

Abstract

Alzheimer's disease (AD) is the leading cause of dementia in elderly people. The etiology of this disease remains a matter of intensive research in many laboratories. We have advanced the idea that disseminated fungal infection contributes to the etiology of AD. Thus, we have demonstrated that fungal proteins and DNA are present in nervous tissue from AD patients. More recently, we have reported that bacterial infections can accompany these mycoses, suggesting that polymicrobial infections exist in AD brains. In the present study, we have examined fungal and bacterial infection in brain tissue from AD patients and control subjects by immunohistochemistry. In addition, we have documented the fungal and bacterial species in brain regions from AD patients and control subjects by next-generation sequencing (NGS). Our results from the analysis of ten AD patients reveal a variety of fungal and bacterial species, although some were more prominent than others. The fungal genera more prevalent in AD patients were Alternaria, Botrytis, Candida, and Malassezia. We also compared these genera with those found in elderly and younger subjects. One of the most prominent genera in control subjects was Fusarium. Principal component analysis clearly indicated that fungi from frontal cortex samples of AD brains clustered together and differed from those of equivalent control subjects. Regarding bacterial infection, the phylum Proteobacteria was the most prominent in both AD patients and controls, followed by Firmicutes, Actinobacteria, and Bacteroides. At the family level, Burkholderiaceae and Staphylococcaceae exhibited higher percentages in AD brains than in control brains. These findings could be of interest to guide targeted antimicrobial therapy for AD patients. Moreover, the variety of microbial species in each patient may constitute a basis for a better understanding of the evolution and severity of clinical symptoms in each patient.

Keywords: Alzheimer’s disease; bacteria and fungal co-infections; fungal infection; infection in aging brain; next generation sequencing; polymicrobial infection.

FIGURE 1

Immunohistochemistry of fungal structures brain sections. Paraffin-embedded brain sections (5 μm) were processed as described (see section “Materials and Methods”). Sections were incubated with rabbit polyclonal anti-C. albicans antibodies (1:100) and an Alexa 488-conjugated donkey anti-rabbit antibody (1:500) (green) and stained with DAPI (blue). A wide file (20× magnification) is shown for the ERH brain section of patient AD11 (A), an elderly subject (C13) (B), and a younger subject (C10) (C). Scale bar is shown in the figure.

FIGURE 2

Nested PCR of fungal DNA from AD frozen tissue. PCR analysis was carried out as described (see section “Materials and Methods”). Schematic representation of fungal rRNA genes (18S, 5.8S and 28S rRNA) and the ITS-1 and ITS-2 regions, including location of the primers employed for the different nested PCRs: primers External 1 employed in the first PCR; primers Internal 1A and Internal 1B employed in the second PCR to amplify ITS-1; primers Internal 2 employed in the second PCR to amplify ITS-2 (A). Agarose gel electrophoresis of the DNA fragments amplified by nested PCR using DNA extracted from frozen FC tissue. PCR analysis of ten AD patients using primers Internal 1B to amplify the ITS-1 region (B). PCR analysis to amplify the ITS-2 region from 10 AD patients using primers Internal 2 (C). PCR analysis of DNA extracted from the samples tested in (B,C) using human β-globin oligonucleotide primers (D). Control–, PCR without DNA. CE, Control of DNA extraction without DNA. C+, DNA extracted from HeLa cells.

FIGURE 3

Distribution of fungal families and genera obtained by NGS of DNA from ten AD patients. Computational analyses of the sequences obtained on the Illumina platform using Qiime classified the data into fungal families and genera. (Upper) Shows the results of fungal families obtained from FC of AD patients. (Lower) Shows the results of fungal genera obtained from FC of AD patients. Asc, Ascomycota; Bas, Basidiomycota; Chy, Chytridiomycota.

FIGURE 4

Nested PCR of fungal DNA from control individuals. PCR analysis was carried out as described (see section “Materials and Methods”). Agarose gel electrophoresis of the DNA fragments amplified by nested PCR. PCR analysis of DNA extracted from frozen ERH tissue of nine controls using primers Internal 1A to amplify the ITS-1 region (A). PCR analysis to amplify region ITS-2 from ERH of nine controls using primers Internal 2 (B). PCR analysis of DNA extracted from the samples tested in (A,B) using human β-globin oligonucleotide primers (C). PCR analysis from FC, ERH, MD, and SC of four controls using primers Internal 1A to amplify the ITS-1 region (D). PCR analysis to amplify ITS-2 region of the same control samples indicated in (D) using primers Internal 2 (E). PCR analysis of DNA extracted from the samples tested in (D,E) using human β-globin oligonucleotide primers (F). Control–, PCR without DNA. CE, control of DNA extraction without DNA. C+, DNA extracted from HeLa cells.

FIGURE 5

Distribution of fungal genera obtained by NGS of DNA from control subjects. Computational analyses of the sequences obtained on the Illumina platform using Qiime classified the data into fungal genera. Results of fungal genera obtained from DNA extracted from nine samples of frozen ERH tissue of controls (C1–C9) and two Superior Frontal Gire controls (C14, C15) and one Parietal cortex control (C16) (A) Results of fungal genera obtained from four CNS regions: FC, ERH, MD, and SC from four controls (B–E). Asc, Ascomycota; Bas, Basidiomycota; Chy, Chytridiomycota.

FIGURE 6

Distribution of fungal genera between AD patients, elderly and younger controls. Median of the percentages of the different genera as indicated in the Figure (A–H).

FIGURE 7

Principal component analysis of AD and control samples: 3D principal component analysis scatter plots of AD patients and controls. Distribution between ten AD patients using FC samples, nine control ERH samples, two Superior Frontal Gire control samples (C14 and C15), and one Parietal cortex control sample (C16) (A). Distribution between ten AD patients using FC samples (plots in blue) and nine ERH samples (plots in red) (B). The UniFrac method was used to calculate this parameter.

FIGURE 8

Immunohistochemistry analysis of bacterial structures in brain sections. Paraffin-embedded ERH sections (5 μm) were processed as indicated (see section “Materials and Methods”) and incubated with mouse monoclonal antibody against peptidoglycan at 1:20 dilution (A–D, K–M,S). Sections were incubated with rabbit polyclonal antibody against C. pneumoniae used at 1:20 dilution (E,F,N–P,T). Sections were incubated with mouse monoclonal antibody against Chlamydia used at 1:10 dilution (G,H,Q,U). Sections were incubated with rabbit polyclonal antibody against Borrelia burgdorferi used at 1:50 dilution (I,J,R,V). In all cases, DAPI staining appears in blue. (A) Patient AD2; (B,C,F) patient AD4; (D) patient AD5; (E) patient AD3; (G,I,J) patient AD7; (H) patient AD10; (K,N) control C11; (L,O) control C12; (M,P) control C13; (Q,R) control C6; (S,T): control C10; (U) control C2, and (V) control C1. (A–J,P–R,T–V) ERH samples; (K) MD sample; (L,N,S) FC samples; (M,O) SC samples. Scale bar: 5 μm for (A–H); 20 μm for (I,J); and 10 μm for (K–V).

FIGURE 9

Distribution of bacteria phyla and orders obtained by NGS of DNA from AD patients. Computational analyses of the sequences obtained on the Illumina platform using Qiime classified the data into bacteria phyla and orders. (Upper) Shows the results of bacteria phyla obtained from ERH samples from 10 AD patients. (Lower) Shows the results of bacterial orders obtained from ERH samples from 10 AD patients.

FIGURE 10

Distribution of bacteria phyla and orders obtained by NGS of DNA from control subjects. Computational analyses of the sequences obtained on the Illumina platform using Qiime classified the data into bacteria phyla and orders. (Upper) Shows the results of bacteria phyla obtained from ERH samples of nine control subjects. (Lower) Shows the results of bacterial orders obtained from ERH samples of nine controls.

FIGURE 11

Distribution of bacteria phyla and families between AD patients and controls. Representation of median percentage of the median values of the percentages of different phyla (A–D). Representation of the median of the percentages of different families (E–J).

FIGURE 12

Principal component analysis of AD and control samples: 3D principal component analysis scatter plots of bacteria from ten AD patients and nine controls. The UniFrac method was used to calculate this parameter.