Biology subjects

Haider, D.

Publications and source records attributed to Haider, D..

4 recordsLinked to original sources

bioRxiv2026

Intubation and mechanical ventilation lead to an extensive expansion of VAP-associated bacterial species in respiratory secretions of ventilated pigs

Ventilator associated pneumonia (VAP) remains one of the most critical nosocomial infections in intensive care medicine. Between 20-50% of ventilated patients develop VAP during treatment, often leading to long term sequelae and death, despite successful therapy of their underlying disease. VAP is typically caused by Gram-negative bacteria that can previously be detected on the mucosal membranes of the oral and upper respiratory tract. To assess and quantify bacterial colonization of the respiratory tract following intubation and ventilation a well-established pig model was used. Methods: After induction of anaesthesia and endotracheal intubation pigs underwent mechanical ventilation continuously for up to three days. To assess bacterial colonization of the upper respiratory tract, samples of respiratory secretions were taken at day 0, 1 and 2 directly above the cuff outside of the endotracheal tube (ETT) and by aspiration of tracheal secretions through the ETT. Quantification of bacteria was carried out by serial dilutions and CFU/ml determinations on sheep blood agar and MacConkey agar plates. Species identification was achieved by 16S-RNA gene sequencing. Results: Respiratory samples of 19 pigs were analysed and showed a sharp increase of predominantly Gram-negative organisms in the respiratory material sampled above the cuff from day 0 to 1. Starting at a median of 7.3 x 103 immediately after intubation numbers increased to a median of 2.0 x 107 Gram-negative bacteria at day 1 and 3.2 x 107 at day 2. Gram-negative species that were detected included Pseudomonas aeruginosa, Acinetobacter baumanii, Enterobacter aerogenes, Klebsiella pneumoniae and other Enterobacteriaceae. Tracheal secretions contained much less bacteria. While no Gram-negative bacteria could be cultured at the beginning of the experiment in 18 of 19 animals, colonization with Gram-negative bacteria gradually increased and was found in 7 of 19 and 9 of 14 animals on day 1 and day 2 respectively. Interestingly, in the vast majority of colonized tracheal secretions the identical Gram-negative species had previously been detected in secretions collected > 24h earlier at the cuff. Conclusion: A massive increase of the concentration of Gram-negative bacteria occurred at 24h of ventilation in mucosal secretions above the cuff. Detected species were the same Gram-negative bacteria that are frequently responsible for human infections, suggesting that especially the accumulation of bacteria in secretions above the cuff may be crucial in the development of VAP.

microbiology↗

bioRxiv2026

Fluoroquinolone-Triggered Prophage Induction in Streptococcus anginosus Reveals Lytic Cycle, CRISPR-Prophage Interplay, and the potential for Cross-Species Horizontal Gene Transfer

Streptococcus anginosus (S. anginosus) has long been considered a commensal of the human microbiome but is increasingly associated with invasive infections and malignant processes. For understanding evolutionary dynamics, it is essential to investigate its mobile genetic elements, such as prophages, which are known to impact virulence, antibiotic resistance, and horizontal gene transfer. While many S. anginosus strains carry prophages, lysogen induction by external stimuli has not been demonstrated, and phage-mediated infection or lysis of this species has not been reported. To analyze the prevalence and diversity of prophages in S. anginosus genomes, we screened 140 clinical isolates by PCR revealing that 31.4% of strains were lysogenic. Correlating these findings with the presence of CRISPR immunity, we observed that S. anginosus strains carrying a CRISPR-Cas type II-A system were less likely to harbor prophages. Using a PCR-based approach, the spontaneous excision of several prophages of S. anginosus could be demonstrated and a fluoroquinolone-triggered prophage induction could successfully be established. Induction by ciprofloxacin and levofloxacin resulted in significant, concentration-dependent phage release and bacterial lysis. Transmission electron microscopy revealed viruses exhibiting the morphology characteristic of siphoviruses. Further analysis of the susceptibility of S. anginosus isolates and other oral and pyogenic streptococci to the isolated S. anginosus phages demonstrated a broad host range and the potential for cross-species horizontal gene transfer. In conclusion, a lytic cycle of S. anginosus phages could be induced, highlighting their functional relevance to pathogenicity and horizontal gene transfer, while demonstrating potential clinical implications of antibiotic-mediated prophage activation.

microbiology↗

bioRxiv2025

Comparison of spatio-temporal dynamics and composition in size-fractionated and unfractionated Northwestern Atlantic microbial communities

Size fractionation is a widely applied approach to target specific microbial size ranges, differentiating between larger particle-associated, and smaller free-living microorganisms. To characterize its impact on microbial diversity and its comparability to unfractionated samples, we analyzed 16 weekly ocean samples across five depths during a spring bloom. A universal marker was used to characterize prokaryotes, eukaryotes and chloroplasts comparing single (0.2 {micro}m) or sequential (3 {micro}m and 0.2 {micro}m) filtration. We analyzed unfractionated, fractionated (small and large fractions) and de-fractionated samples. The particle-associated fraction defines the most different community from the other fractions, and combining size fractions (de-fractionating) before or after sequencing produces a community that is most similar to unfractionated samples in terms of composition, and richness dynamics with the exception of very rare taxa. Between 75% and 97% of features are shared, but some discrepancies in relative abundances were unresolved, including for some lineages of free-living Proteobacteria like OM43. The richness trends were consistent, and ANCOM detected at most one significantly different feature between fractionated, and de-fractionated samples, highlighting the similarity in community composition and temporal dynamics between the sets.

molecular biology↗

bioRxiv2024

The stress of carrying CRISPR-Cas

Streptococcus anginosus (S. anginosus) is a commensal that can cause severe invasive bacterial infections. A considerable percentage of S. anginosus strains harbor CRISPR-Cas systems, which apart from being a bacterial immunity system can play an important role regarding the adaptation to environmental stress. The functionality of S. anginosus CRISPR-Cas systems has previously not been investigated. To address this, we created a set of deletion mutants in the CRISPR-Cas type II-A system of the S. anginosus SK52 type strain, targeting the nuclease Cas9 and the CRISPR array. Testing these strains in a plasmid clearance assay, we were able to confirm CRISPR-Cas activity. Furthermore, the role of the S. anginosus CRISPR-Cas system was investigated under various stress conditions such as UV light, hydrogen peroxide exposure, and high-temperatures in wildtype S. anginosus and CRISPR-Cas mutant strains. Under these conditions, survival was significantly lower in strains carrying cas9. Bacterial growth and metabolic activity in Alamar blue assays was also negatively affected by the presence of cas9 in S. anginosus. In summary we found that the presence of a functional CRISPR-Cas system in S. anginosus leads to measurable metabolic and fitness costs for the wildtype strain. Carrying cas9 was associated with an impaired stress response in our experiments and may thus explain, why many strains of this species lack CRISPR-Cas. Author SummaryThe bacterial immunity system CRIPRS-Cas provides protection against invading foreign genetic material. Despite this obvious advantage only about 50% of bacteria carry CRISPR-Cas. To investigate the CRISPR system of Streptococcus anginosus, which can cause serious bacterial infections and has recently been linked to gastric cancer, we created a set of mutants in different loci of the CRISPR system. Exposing these mutants to stress through UV-light, hydrogen peroxide and high temperatures, we could show that carrying the CRISPR nuclease gene Cas9 is associated with impaired survival under harsh conditions. Strains lacking the nuclease gene had a better growth and higher metabolic activity than the wildtype strain. In summary we found that the presence of a functional CRISPR-Cas system in S. anginosus leads to considerable metabolic and fitness costs. Carrying cas9 was associated with an impaired stress response in our experiments and may thus explain, why many strains of this species lack CRISPR-Cas.

microbiology↗