Staff Publications

Streptococcus suis is a porcine pathogen, causing severe invasive infections. S. suis serotype 9 is increasingly causing disease in Dutch and Chinese pig herds, but it is unknown whether all serotype 9 isolates are equally virulent and markers that can identify virulent strains are not available. Therefore, discrimination between virulent isolates and carriage isolates typically not associated with disease, is currently not possible. We collected tonsillar S. suis isolates from 6 herds not previously diagnosed with S. suis infections, and clinical S. suis isolates of previously diseased pigs. We confirmed the virulence of a virulent type strain and one representative clinical isolate, and the lack of virulence of two carriage isolates, in a pig infection model. Phylogenetic analysis of whole genome sequences of 124 isolates resulted in 10 groups, of which two were almost uniquely populated by clinical isolates. The population structure of S. suis serotype 9 appears highly diverse. However, analysis of the capsule loci sequences showed variation in a single region which fully correlated with a virulent genotype. Transmission electron microscopy suggested differences in capsule thickness between carriage and clinical genotypes. In conclusion, we found that that the S. suis serotype 9 population in the Netherlands is diverse. A distinct virulence-associated lineage was identified and could be discriminated based on the capsule locus sequence. Whilst the difference in virulence cannot be directly attributed to the DNA sequence, the correlation of capsule locus sequence with virulence could be used in the development of diagnostic tests to identify potential virulent S. suis serotype 9 in pigs.

Clostridium difficile infections (CDI) are a major cause of antibiotic-associated diarrhea. It is hypothesized that CDI develops due to the antibiotic-induced disruption of the intestinal microbial community structure, which allows C. difficile to flourish. Here, we pre-treated weaned pigs with the antibiotics Clindamycin or Ciprofloxacin for 1 day, and subsequently inoculated them with a human and pig enteropathogenic C. difficile strain 078 spores. Body temperature, clinical signs of disease, and the fecal microbiome were monitored daily for 15 days. Clindamycin had a stronger effect on the pigs than Ciprofloxacin, resulting in drastic shifts in the fecal microbiome, decreases in microbial diversity and significant increases in body temperature, even in the absence of C. difficile. Fecal shedding of C. difficile was detectable for 3 and 9 days in Ciprofloxacin and Clindamycin treated pigs inoculated with C. difficile, respectively, and in both cases decreased cell proliferation rates were detected in colon tissue. The timing of C. difficile shedding coincided with the decrease in a large cluster of Firmicutes following Clindamycin treatment, a pattern which was also independent of C. difficile inoculation. The observed community patterns suggest that successional dynamics following antibiotic treatment facilitate C. difficile establishment. The similarities between the microbiome responses observed in our study and those previously reported in CDI-infected humans further support the utility of adult pigs as models for the study of CDI.

Scope: Akkermansia muciniphila (A. muciniphila) is an intestinal commensal with anti-inflammatory properties both in the intestine and other organs. The aim is to investigate the effects of oral administration of A. muciniphila on lipid metabolism, immunity, and cuff-induced neointima formation in hyperlipidemic APOE*3-Leiden (E3L).CETP mice. Methods and results: Hyperlipidemic male E3L.CETP mice are daily treated with 2 × 10⁸ CFU A. muciniphila by oral gavage for 4 weeks and the effects are determined on plasma lipid levels, immune parameters, and cuff-induced neointima formation and composition. A. muciniphila administration lowers body weight and plasma total cholesterol and triglycerides levels. A. muciniphila influences the immune cell composition in mesenteric lymph nodes, as evident from an increased total B cell population, while reducing the total T cell and neutrophil populations. Importantly, A. muciniphila reduces the expression of the activation markers MHCII on dendritic cells and CD86 on B cells. A. muciniphila also increases whole blood ex vivo lipopolysaccharide-stimulated IL-10 release. Finally, although treatment with A. muciniphila improves lipid metabolism and immunity, it does not affect neointima formation or composition. Conclusions: Four weeks of treatment with A. muciniphila exerts lipid-lowering and immunomodulatory effects, which are insufficient to inhibit neointima formation in hyperlipidemic E3L.CETP mice.

There is a need to develop diagnostic and analytical tools that allow noninvasive monitoring of bacterial growth and dissemination in vivo. For such cell-tracking studies to hold translational value to controlled human infections, in which volunteers are experimentally colonized, they should not require genetic modification, and they should allow tracking over a number of replication cycles. To gauge if an antimicrobial peptide tracer, ^99m Tc-UBI _29-41 -Cy5, which contains both a fluorescent and a radioactive moiety, could be used for such in vivo bacterial tracking, we performed longitudinal imaging of a thigh-muscle infection with ^99m Tc-UBI _29-41 -Cy5-labeled Staphylococcus aureus. Mice were imaged using SPECT and fluorescence-imaging modalities at various intervals during a 28 h period. Biodistribution analyses were performed to quantitate radioactivity in the abscess and other tissues. SPECT and fluorescence imaging in mice showed clear retention of the ^99m Tc-UBI _29-41 -Cy5-labeled bacteria following inoculation in the thigh muscle. Despite bacterial replication, the signal intensity in the abscess only modestly decreased within a 28 h period: 52% of the total injected radioactivity per gram of tissue (%ID/g) at 4 h postinfection (pi) versus 44%ID/g at 28 h pi (15% decrease). After inoculation, a portion of the bacteria disseminated from the abscess, and S. aureus cultures were obtained from radioactive urine samples. Bacterial staining with ^99m Tc-UBI _29-41 -Cy5 allowed noninvasive bacterial-cell tracking during a 28 h period. Given the versatility of the presented bacterial-tracking method, we believe that this concept could pave the way for precise imaging capabilities during controlled-human-infection studies.

Background: Today, both radioactive SPECT and PET imaging radiopharmaceuticals are used for clinical diagnosis of bacterial infections. Due to the possible applications in image-guided surgery, fluorescent imaging of infections has gained interest. We here present the highlights and recent developments in the use of fluorescence imaging for bacterial infections. In this overview, we also include the latest developments in multimodal bacterial imaging strategies that combine radioactive and fluorescent imaging. Based on this literature, we present our future perspectives for the field including the translational potential. Methods: In the current review, we complement earlier reports with the most recent fluorescent and multimodal radiopharmaceuticals for bacterial infection imaging. Where possible, in this review, the chemical structure of the compounds and clinical images were shown. Results: A total of 35 out of 77 original articles on pre-clinical and clinical imaging of bacterial infections with fluorescent tracers and multimodality radiopharmaceuticals were included for reviewing. Conclusion: In our view, the highest translational potential lies with compounds that are based on targeting vectors that are specific for bacteria: e.g., fluorescently labelled UBI _29–41 , polymyxin B, vancomycin, ZnDPA and a M. tuberculosis-specific β-lactamase-cleavable linker CNIP800. Multimodal concepts using dually labelled UBI _29–41 , vancomycin, and ZnDPA help connect optical imaging to the more traditional use of radiopharmaceuticals in infectious diseases. Multimodal bacterial imaging is a promising strategy not only to diagnose bacterial infections but also to evaluate the effectivity of surgical treatment for infections.