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BackgroundKlebsiella pneumoniae (Kp) is an ecologically generalist bacterium but also an opportunistic pathogen responsible for hospital-acquired infections and a major contributor to the global burden of antimicrobial resistance. In the last decades, few advances have been made in the use of virulent phages as alternative or complement to antibiotics to treat Kp infections. The efficiency of phages relies on their ability to recognize and attach to the bacterial surface structure, and in the case of Kp, capsule (K) is the main surface structure. However, Kp capsule is highly polymorphic and the majority of classically isolated phages are specific for unique K-types, limiting therapy prospects. In this study, we demonstrate the feasibility of an innovative strategy consisting in isolating phages that target capsule-deficient mutant Kp strains, and compare such phages with anti-capsulated cells phages phylogenetically and through in vitro and in vivo experiments.MethodsWe isolated 27 phages using 7 capsule-deficient Kp strains as hosts (anti-Kd phages), and 41 phages against 7 wild-type (wt) Kp strains (anti-K phages). We evaluated and compared phenotypically and genotypically their host range, resistance emergence and selected mutations and in-vivo activity.ResultsIn vitro, anti-Kd phages showed a broader host-range, with most phages being able to infect non-capsulated mutants of multiple sublineages and O-antigen locus types. Besides, the emergence of bacterial subpopulations non-susceptible to anti-Kd phages was slower when compared to anti-K phages and with a different range of genomic differences. One anti-Kd phage (mtp5) was shown to infect non-capsulated Kp strains belonging to 10 of the 12 known O-antigen types. Moreover, this phage was able to replicate in the gut of mice colonised with the wt (capsulated) parent strain.ConclusionsThis work demonstrates the potential value of an anti-Klebsiella phage isolation strategy that addresses the issue of narrow host-range of anti-K phages. Anti Kd-phages may be active in infection sites where capsule expression is intermittent or repressed, or in combination with anti-K phages, which often induce loss of capsule escape mutants.

Feed supplements such as Saccharomyces cerevisiae fermentation products (SCFP) alter immune responses in horses. The purpose of this study was to analyze whether a prebiotic activity of the SCFP alters the gut microbiome in horses. Racehorses were fed either SCFP (Olimond BB, OLI, n = 6) or placebo pellets (PLA, n = 5) for 43 days. Fecal microbiota analysis was performed using 16S rRNA gene sequencing. The numbers and function of circulating immune cell subpopulations were analyzed by flow cytometry. SCFP supplementation resulted in non-consistent differences in fecal microbiota between the PLA and OLI during the feeding period. Rather, the individual animal had the highest impact on fecal microbiota composition. OLI and PLA horses displayed the same changes in numbers of blood leukocyte subpopulations over time. One day after a booster vaccination against equine influenza during the feeding period, the alpha diversity of fecal microbiota of PLA horses was significantly higher compared to OLI horses. This suggests that SCFP feeding altered the vaccination-induced spectrum of released mediators, potentially affecting gut microbiota. The overall non-consistent findings argue against a strong prebiotic effect of Olimond BB on the microbiota in racehorses. Fecal microbiota differences between the groups were also noticed outside the feeding period and, hence, are most likely not caused by the SCFP additive.

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Summary Gut colonization with multidrug-resistant (MDR) bacteria enhances the risk of bloodstream infections in susceptible individuals. We demonstrate highly variable degrees of ex vivo colonization resistance against a carbapenem-resistant Klebsiella pneumoniae strain in human feces samples and subsequently isolate diverse K. oxytoca strains from protected donors. Several of these K. oxytoca strains reduce gut colonization of MDR K. pneumoniae strains in antibiotic-treated and gnotobiotic mouse models. Comparative analysis of K. oxytoca strains coupled with CRISPR-Cas9-mediated deletion of casA, a protein essential for utilization of selected beta-glucosides, identified competition for specific carbohydrates as key in promoting colonization resistance. In addition to direct competition between K. oxytoca and K. pneumoniae, cooperation with additional commensals is required to reestablish full colonization resistance and gut decolonization. Finally, humanized microbiota mice generated from K. pneumoniae-susceptible donors are protected by K. oxytoca administration, demonstrating the potential of commensal K. oxytoca strains as next-generation probiotics.

The composition of the intestinal microbiota influences the outcome of enteric infections in human and mice. However, the role of specific members and their metabolites contributing to disease severity is largely unknown. Using isogenic mouse lines harboring distinct microbiota communities, we observed highly variable disease kinetics of enteric Citrobacter rodentium colonization after infection. Transfer of communities from susceptible and resistant mice into germ-free mice verified that the varying susceptibilities are determined by microbiota composition. The strongest differences in colonization were observed in the cecum and could be maintained in vitro by coculturing cecal bacteria with C. rodentium. Cohousing of animals as well as the transfer of cultivable bacteria from resistant to susceptible mice led to variable outcomes in the recipient mice. Microbiome analysis revealed that a higher abundance of butyrate-producing bacteria was associated with the resistant phenotype. Quantification of short-chain fatty acid (SCFA) levels before and after infection revealed increased concentrations of acetate, butyrate and propionate in mice with delayed colonization. Addition of physiological concentrations of butyrate, but not of acetate and/or propionate strongly impaired growth of C. rodentium in vitro. In vivo supplementation of susceptible, antibiotic-treated and germ-free mice with butyrate led to the same level of protection, notably only when cecal butyrate concentration reached a concentration higher than 50 nmol/mg indicating a critical threshold for protection. In the recent years, commensal-derived primary and secondary bacterial metabolites emerged as potent modulators of hosts susceptibility to infection. Our results provide evidence that variations in SCFA production in mice fed fibre-rich chow-based diets modulate susceptibility to colonization with Enterobacteriaceae not only in antibiotic-disturbed ecosystems but even in undisturbed microbial communities. These findings emphasise the need for microbiota normalization across laboratory mouse lines for infection experiments with the model-pathogen C. rodentium independent of investigations of diet and antibiotic usage., Author summary The distinct composition of the gut microbiota in each individual results in variable metabolic activity and output of these communities, which influences the host, including resistance to enteric pathogens. Lack of reproducibility in biomedical research is nowadays frequently attributed to the microbiota, but little is known about which specific members and metabolites contribute to disease severity. Here, we use genetically identical mouse lines with variable microbiota compositions on a standardized diet and observed highly variable colonization with the enteric pathogen Citrobacter rodentium without antibiotics intervention. We found the same differences in formerly germ-free animals harbouring the respective donors microbiota and also in vitro by coculturing cecal bacteria from resistant and susceptible animals with C. rodentium showing that the phenotype is fully dependent on differences in the microbiota. We analysed the microbiome composition and found a higher abundance of butyrate-producing bacteria as well as increased levels of butyrate in resistant mice. By supplementation of susceptible and germ-free animals with butyrate, we could significantly lower the levels of colonization highlighting that commensal-derived primary and secondary bacterial metabolites are highly variable between laboratory animals from different vendors and are potent modulators of hosts susceptibility to infection with C. rodentium.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is one of the few oncogenic human viruses known to date. Its large genome encodes more than 85 proteins and includes both unique viral proteins as well as proteins conserved amongst herpesviruses. KSHV ORF20 is a member of the herpesviral core UL24 family, but the function of ORF20 and its role in the viral life cycle is not well understood. ORF20 encodes three largely uncharacterized isoforms, which we found were localized predominantly in the nuclei and nucleoli. Quantitative affinity purification coupled to mass spectrometry (q-AP-MS) identified numerous specific interacting partners of ORF20, including ribosomal proteins and the interferon-stimulated gene product (ISG) oligoadenylate synthetase-like protein (OASL). Both endogenous and transiently transfected OASL co-immunoprecipitated with ORF20, and this interaction was conserved among all ORF20 isoforms and multiple ORF20 homologs of the UL24 family in other herpesviruses. Characterization of OASL interacting partners by q-AP-MS identified a very similar interactome to that of ORF20. Both ORF20 and OASL copurified with 40S and 60S ribosomal subunits, and when they were co-expressed, they associated with polysomes. Although ORF20 did not have a global effect on translation, ORF20 enhanced RIG-I induced expression of endogenous OASL in an IRF3-dependent but IFNAR-independent manner. OASL has been characterized as an ISG with antiviral activity against some viruses, but its role for gammaherpesviruses was unknown. We show that OASL and ORF20 mRNA expression were induced early after reactivation of latently infected HuARLT-rKSHV.219 cells. Intriguingly, we found that OASL enhanced infection of KSHV. During infection with a KSHV ORF20stop mutant, however, OASL-dependent enhancement of infectivity was lost. Our data have characterized the interaction of ORF20 with OASL and suggest ORF20 usurps the function of OASL to benefit KSHV infection., Author summary The herpesviruses are a family of large double-stranded DNA viruses that cause a variety of illnesses from chicken pox to cancer. Kaposi’s sarcoma-associated herpesvirus (KSHV) is a cancer-causing herpesvirus and can lead to development of Kaposi’s sarcoma, a major form of cancer in HIV-positive patients. As for all herpesviruses, infection with KSHV is lifelong. Exactly how KSHV initiates and maintains its infection is still not well understood, but it must manipulate the host cell to establish favorable conditions. Likewise, the host has developed a complicated system to fight off invaders, which includes the production of interferon-stimulated gene products. We have now found that KSHV exploits one such host cell protein, the oligoadenylate synthetase-like protein (OASL). Rather than OASL acting as an antiviral protein as it does during many other viral infections, KSHV appears to have found a way to utilize OASL for its own benefit. The KSHV protein ORF20 interacts with OASL, they co-localize in nucleoli, and both ORF20 and OASL associate and purify with components of the cellular translational machinery. This may help viral infection by selectively controlling protein production.