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Molecular Characterization of Microbiota in Cerebrospinal Fluid From Patients With CSF Shunt Infections Using Whole Genome Amplification Followed by Shotgun Sequencing.

ABSTRACT: Understanding the etiology of cerebrospinal fluid (CSF) shunt infections and reinfections requires detailed characterization of associated microorganisms. Traditionally, identification of bacteria present in the CSF has relied on culture methods, but recent studies have used high throughput sequencing of 16S rRNA genes. Here we evaluated the method of shotgun DNA sequencing for its potential to provide additional genomic information. CSF samples were collected from 3 patients near the beginning and end of each of 2 infection episodes. Extracted total DNA was sequenced by: (1) whole genome amplification followed by shotgun sequencing (WGA) and (2) high-throughput sequencing of the 16S rRNA V4 region (16S). Taxonomic assignments of sequences from WGA and 16S were compared with one another and with conventional microbiological cultures. While classification of bacteria was consistent among the 3 approaches, WGA provided additional insights into sample microbiological composition, such as showing relative abundances of microbial versus human DNA, identifying samples of questionable quality, and detecting significant viral load in some samples. One sample yielded sufficient non-human reads to allow assembly of a high-quality Staphylococcus epidermidis genome, denoted CLIMB1, which we characterized in terms of its MLST profile, gene complement (including putative antimicrobial resistance genes), and similarity to other annotated S. epidermidis genomes. Our results demonstrate that WGA directly applied to CSF is a valuable tool for the identification and genomic characterization of dominant microorganisms in CSF shunt infections, which can facilitate molecular approaches for the development of better diagnostic and treatment methods.

SUBMITTER: Hodor P

PROVIDER: S-EPMC8417900 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundNearly 20% of patients with cerebrospinal fluid (CSF) shunt infection develop reinfection. It is unclear whether reinfections are caused by an organism previously present or are independent infection events.ObjectiveWe used bacterial culture and high throughput sequencing (HTS) of 16S ribosomal RNA (rRNA) genes to identify bacteria present in serial CSF samples obtained from children who failed CSF shunt infection treatment. We hypothesized that organisms that persist in CSF despite treatment would be detected upon reinfection.Design/methodsSerial CSF samples were obtained from 6 patients, 5 with 2 infections and 1 with 3 infections; the study was limited to those for which CSF samples were available from the end of infection and beginning of reinfection. Amplicons of the 16S rRNA gene V4 region were sequenced. Taxonomic assignments of V4 sequences were compared with bacterial species identified in culture.ResultsSeven infection dyads averaging 13.5 samples per infection were analyzed. A median of 8 taxa [interquartile range (IQR) 5-10] were observed in the first samples from reinfection using HTS. Conventional culture correlated with high abundance of an organism by HTS in all but 1 infection. In 6 of 7 infection dyads, organisms identified by culture at reinfection were detected by HTS of culture-negative samples at the end of the previous infection. The median Chao-Jaccard abundance-based similarity index for matched infection pairs at end of infection and beginning of reinfection was 0.57 (IQR 0.07-0.87) compared to that for unmatched pairs of 0.40 (IQR 0.10-0.60) [p = 0.46].Conclusion(s)HTS results were generally consistent with culture-based methods in CSF shunt infection and reinfection, and may detect organisms missed by culture at the end of infection treatment but detected by culture at reinfection. However, the CSF microbiota did not correlate more closely within patients at the end of infection and beginning of reinfection than between any two unrelated infections. We cannot reject the hypothesis that sequential infections were independent.

Project description:BackgroundApproximately 30% of cerebrospinal fluid (CSF) shunt systems for hydrocephalus fail within the first year and 98% of all patients will have shunt failure in their lifetime. Obstruction remains the most common reason for shunt failure. Previous evidence suggests elevated pro-inflammatory cytokines in CSF are associated with worsening clinical outcomes in neuroinflammatory diseases. The aim of this study was to determine whether cytokines and matrix metalloproteinases (MMPs) contribute towards shunt failure in hydrocephalus.MethodsUsing multiplex ELISA, this study examined shunt failure through the CSF protein concentration profiles of select pro-inflammatory and anti-inflammatory cytokines, as well as select MMPs. Interdependencies such as the past number of previous revisions, length of time implanted, patient age, and obstruction or non-obstruction revision were examined. The pro-inflammatory cytokines were IL-1?, IL-2, IL-5, IL-6, IL-8, IL-12, IL-17, TNF-?, GM-CSF, IFN-?. The anti-inflammatory cytokines were IL-4 and IL-10, and the MMPs were MMP-2, MMP-3, MMP-7, MMP-9. Protein concentration is reported as pg/mL for each analyte.ResultsPatient CSF was obtained at the time of shunt revision operation; all pediatric (<?18), totaling n?=?38. IL-10, IL-6, IL-8 and MMP-7 demonstrated significantly increased concentrations in patient CSF for the non-obstructed subgroup. Etiological examination revealed IL-6 was increased in both obstructed and non-obstructed cases for PHH and congenital hydrocephalic patients, while IL-8 was higher only in PHH patients. In terms of number of past revisions, IL-10, IL-6, IL-8, MMP-7 and MMP-9 progressively increased from zero to two past revisions and then remained low for subsequent revisions. This presentation was notably absent in the obstruction subgroup. Shunts implanted for three months or less showed significantly increased concentrations of IL-6, IL-8, and MMP-7 in the obstruction subgroup. Lastly, only patients aged six months or less presented with significantly increased concentration of IL-8 and MMP-7.ConclusionNon-obstructive cases are reported here to accompany significantly higher CSF cytokine and MMP protein levels compared to obstructive cases for IL-10, IL-6, IL-8, MMP-7 and MMP-9. A closer examination of the definition of obstruction and the role neuroinflammation plays in creating shunt obstruction in hydrocephalic patients is suggested.

Project description:BackgroundCerebrospinal fluid shunt infection can be recalcitrant. Recurrence is common despite appropriate therapy for the pathogens identified by culture. Improved diagnostic and therapeutic approaches are required, and culture-independent molecular approaches to cerebrospinal fluid shunt infections have not been described.ObjectivesTo identify the bacteria and fungi present in cerebrospinal fluid from children with cerebrospinal fluid shunt infection using a high-throughput sequencing approach, and to compare those results to those from negative controls and conventional culture.MethodsThis descriptive study included eight children ≤18 years old undergoing treatment for culture-identified cerebrospinal fluid shunt infection. After routine aerobic culture of each cerebrospinal fluid sample, deoxyribonucleic acid (DNA) extraction was followed by amplification of the bacterial 16S rRNA gene and the fungal ITS DNA region tag-encoded FLX-Titanium amplicon pyrosequencing and microbial phylogenetic analysis.ResultsThe microbiota analyses for the initial cerebrospinal fluid samples from all eight infections identified a variety of bacteria and fungi, many of which did not grow in conventional culture. Detection by conventional culture did not predict the relative abundance of an organism by pyrosequencing, but in all cases, at least one bacterial taxon was detected by both conventional culture and pyrosequencing. Individual bacterial species fluctuated in relative abundance but remained above the limits of detection during infection treatment.ConclusionsNumerous bacterial and fungal organisms were detected in these cerebrospinal fluid shunt infections, even during and after treatment, indicating diverse and recalcitrant shunt microbiota. In evaluating cerebrospinal fluid shunt infection, fungal and anaerobic bacterial cultures should be considered in addition to aerobic bacterial cultures, and culture-independent approaches offer a promising alternative diagnostic approach. More effective treatment of cerebrospinal fluid shunt infections is needed to reduce unacceptably high rates of reinfection, and this work suggests that one effective strategy may be reduction of the diverse microbiota present in infection.

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Molecular Characterization of Microbiota in Cerebrospinal Fluid From Patients With CSF Shunt Infections Using Whole Genome Amplification Followed by Shotgun Sequencing.

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