Succession in the caecal microbiota of developing broilers colonised by extended-spectrum β-lactamase-producing Escherichia coli

Background Broilers are among the most common and dense poultry production systems, where antimicrobials have been used extensively to promote animal health and performance. The continuous usage of antimicrobials has contributed to the appearance of resistant bacteria, such as extended-spectrum β-lactamase-producing Escherichia coli (ESBL-Ec). Here, we studied the ESBL-Ec prevalence and successional dynamics of the caecal microbiota of developing broilers in a commercial flock during their production life cycle (0–35 days). Broilers were categorised as ESBL-Ec colonised (ESBL-Ec+) or ESBL-Ec non-colonised (ESBL-Ec−) by selective culturing. Using 16S rRNA gene sequencing, we i. compared the richness, evenness and composition of the caecal microbiota of both broilers’ groups and ii. assessed the combined role of age and ESBL-Ec status on the broilers’ caecal microbiota. Results From day two, we observed an increasing linear trend in the proportions of ESBL-Ec throughout the broilers' production life cycle, X2 (1, N = 12) = 28.4, p < 0.001. Over time, the caecal microbiota richness was consistently higher in ESBL-Ec− broilers, but significant differences between both broilers’ groups were found exclusively on day three (Wilcoxon rank-sum test, p = 0.016). Bray–Curtis distance-based RDA (BC-dbRDA) showed no explanatory power of ESBL-Ec status, while age explained 14% of the compositional variation of the caecal microbiota, F (2, 66) = 6.47, p = 0.001. Conclusions This study assessed the role of ESBL-Ec in the successional dynamics of the caecal microbiota in developing broilers and showed that the presence of ESBL-Ec is associated with mild but consistent reductions in alpha diversity and with transient bacterial compositional differences. We also reported the clonal spread of ESBL-Ec and pointed to the farm environment as a likely source for ESBLs

Succession in the caecal microbiota of developing broilers colonised by extended-spectrum β-lactamase-producing Escherichia coli

Background Broilers are among the most common and dense poultry production systems, where antimicrobials have been used extensively to promote animal health and performance. The continuous usage of antimicrobials has contributed to the appearance of resistant bacteria, such as extended-spectrum β-lactamase-producing Escherichia coli (ESBL-Ec). Here, we studied the ESBL-Ec prevalence and successional dynamics of the caecal microbiota of developing broilers in a commercial flock during their production life cycle (0–35 days). Broilers were categorised as ESBL-Ec colonised (ESBL-Ec+) or ESBL-Ec non-colonised (ESBL-Ec−) by selective culturing. Using 16S rRNA gene sequencing, we i. compared the richness, evenness and composition of the caecal microbiota of both broilers’ groups and ii. assessed the combined role of age and ESBL-Ec status on the broilers’ caecal microbiota. Results From day two, we observed an increasing linear trend in the proportions of ESBL-Ec throughout the broilers' production life cycle, X2 (1, N = 12) = 28.4, p < 0.001. Over time, the caecal microbiota richness was consistently higher in ESBL-Ec− broilers, but significant differences between both broilers’ groups were found exclusively on day three (Wilcoxon rank-sum test, p = 0.016). Bray–Curtis distance-based RDA (BC-dbRDA) showed no explanatory power of ESBL-Ec status, while age explained 14% of the compositional variation of the caecal microbiota, F (2, 66) = 6.47, p = 0.001. Conclusions This study assessed the role of ESBL-Ec in the successional dynamics of the caecal microbiota in developing broilers and showed that the presence of ESBL-Ec is associated with mild but consistent reductions in alpha diversity and with transient bacterial compositional differences. We also reported the clonal spread of ESBL-Ec and pointed to the farm environment as a likely source for ESBLs

Succession in the caecal microbiota of developing broilers colonised by extended-spectrum β-lactamase-producing Escherichia coli

Background: Broilers are among the most common and dense poultry production systems, where antimicrobials have been used extensively to promote animal health and performance. The continuous usage of antimicrobials has contributed to the appearance of resistant bacteria, such as extended-spectrum β-lactamase-producing Escherichia coli (ESBL-Ec). Here, we studied the ESBL-Ec prevalence and successional dynamics of the caecal microbiota of developing broilers in a commercial flock during their production life cycle (0–35 days). Broilers were categorised as ESBL-Ec colonised (ESBL-Ec+) or ESBL-Ec non-colonised (ESBL-Ec−) by selective culturing. Using 16S rRNA gene sequencing, we i. compared the richness, evenness and composition of the caecal microbiota of both broilers’ groups and ii. assessed the combined role of age and ESBL-Ec status on the broilers’ caecal microbiota.Results: From day two, we observed an increasing linear trend in the proportions of ESBL-Ec throughout the broilers' production life cycle, X2 (1, N = 12) = 28.4, p < 0.001. Over time, the caecal microbiota richness was consistently higher in ESBL-Ec− broilers, but significant differences between both broilers’ groups were found exclusively on day three (Wilcoxon rank-sum test, p = 0.016). Bray–Curtis distance-based RDA (BC-dbRDA) showed no explanatory power of ESBL-Ec status, while age explained 14% of the compositional variation of the caecal microbiota, F (2, 66) = 6.47, p = 0.001.Conclusions: This study assessed the role of ESBL-Ec in the successional dynamics of the caecal microbiota in developing broilers and showed that the presence of ESBL-Ec is associated with mild but consistent reductions in alpha diversity and with transient bacterial compositional differences. We also reported the clonal spread of ESBL-Ec and pointed to the farm environment as a likely source for ESBLs

Incomplete bunyavirus particles can cooperatively support virus infection and spread

Bunyaviruses lack a specific mechanism to ensure the incorporation of a complete set of genome segments into each virion, explaining the generation of incomplete virus particles lacking one or more genome segments. Such incomplete virus particles, which may represent the majority of particles produced, are generally considered to interfere with virus infection and spread. Using the three-segmented arthropod-borne Rift Valley fever virus as a model bunyavirus, we here show that two distinct incomplete virus particle populations unable to spread autonomously are able to efficiently complement each other in both mammalian and insect cells following co-infection. We further show that complementing incomplete virus particles can co-infect mosquitoes, resulting in the reconstitution of infectious virus that is able to disseminate to the mosquito salivary glands. Computational models of infection dynamics predict that incomplete virus particles can positively impact virus spread over a wide range of conditions, with the strongest effect at intermediate multiplicities of infection. Our findings suggest that incomplete particles may play a significant role in within-host spread and between-host transmission, reminiscent of the infection cycle of multipartite viruses

Source data underlying Figs 1C, 2G, 3C, 4D and 6B.

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Primers for cDNA synthesis of viral genome segments.

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Visualization of viral genome segments in RVFV-infected cells and in immobilized RVFV virions from virus stocks and co-infection supernatants.

(A) Schematic representation of the single-molecule viral RNA FISH-immunofluorescence method used to visualize the intracellular viral ribonucleoprotein (vRNP) composition of infected cells and the intravirion genomic composition of virus stocks and culture supernatants (illustration based on [22]). (B) BSR-T7/5 cells were mock infected, infected exclusively with one population of incomplete particles (MOI 1), or simultaneously infected with iRVFV-SL-eGFP and iRVFV-ML (MOI 1 for each virus), and fixed at 16 h post-infection. Cells infected with a three-segmented RVFV-Clone 13 (MOI 1) were used as positive control. (C) RVFV virions from incomplete particle stocks and supernatants of co-infected BSR-T7/5 and C6/36 cells were immobilized on coverglass by incubation for 5 h at 28°C. The S segment (N gene, red), M segment (polyprotein gene, blue), and L segment (RdRp gene, yellow) were hybridized using probe sets labeled with CAL Fluor Red 610, Quasar 670, and Quasar 570, respectively. RVFV particles (green) were detected with antibody 4-D4 [47] targeting the Gn glycoprotein in combination with Alexa Fluor 488–conjugated secondary antibodies. Cell nuclei (cyan) were visualized with DAPI. Merge images show the overlay of five (for cells) or four (for virions) individual channels. Individual spots, each representing either a single vRNP or a virus particle, were detected and assessed for co-localization in ImageJ with the plugin ComDet. Colored circles display the spots detected in each channel and their co-localization in the merge image. Scale bars, 10 μm (B), 5 μm (C). Illustration in Fig 5A was created with BioRender.com.</p

Co-infection of mammalian and insect cells with recombinant three-segmented RVFV reporter viruses.

(A) Schematic representation of the T7 polymerase-based reverse genetics system. Fluorescently marked variants of RVFV were generated by simultaneous transfection of BSR-T7/5 cells with the transcription plasmids pUC57_S, pUC57_M, and pUC57_L encoding the RVFV-35/74 S, M and L genome segments, respectively, in antigenomic-sense orientation. The pUC57_S plasmid additionally encoded for either eGFP or mCherry2 in place of the NSs gene. (B) Direct fluorescence detection of BSR-T7/5 cells infected with either RVFV-eGFP (green) or RVFV-mCherry2 (red) (MOI 0.5) at 24 h post-infection. (C) Growth kinetics of RVFV-eGFP and RVFV-mCherry2 after infection of BSR-T7/5 cells at an MOI of 0.01. Virus titers were determined with an end-point dilution assay (fluorescence microscopy readout). Dots represent means of biological replicates (n = 3) at each time point, and the shaded area represents the standard deviation. The dashed line indicates the limit of detection (101.8 TCID50/mL). Source data are provided in S1 Data. (D) Schematic representation of the simultaneous infection of mammalian (BSR-T7/5) or insect (C6/36) cells with RVFV-eGFP and RVFV-mCherry2. (E) Direct fluorescence detection of BSR-T7/5 and C6/36 cells co-infected with RVFV-eGFP and RVFV-mCherry2 (MOI 0.5 for each virus) at 24 h (BSR-T7/5) or 72 h (C6/36) post-infection. Inset images are magnifications of a region of interest (indicated as a dashed box). Co-infected cells co-express eGFP (green) and mCherry2 (red) and thus appear yellow. Scale bars, 200 μm (inset images 100 μm). Illustrations in Fig 1A and 1D were created with BioRender.com.</p

Modeling virus spread: simple sensitivity analysis.

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