Chicken faecal microbiota and disturbances induced by single or repeated therapy with tetracycline and streptomycin

BACKGROUND: In this study, we characterised the microbiota present in the faeces of 15- and 46-week-old egg laying hens before and after tetracycline or streptomycin therapy. In the first experiment, the layers were subjected to 7 days of therapy. In the second experiment, the hens were subjected to two days of therapy, which was repeated for an additional two days after 12 days of antibiotic withdrawal. This enabled us to characterise dynamics of the changes after antibiotic administration and withdrawal, and to identify genera repeatedly resistant to tetracycline and streptomycin. RESULTS: Real-time PCRs specific for Enterobacteriales, Lactobacillales, Clostridiales and Bifidobacteriales showed that changes in the microbiota in response to antibiotic therapy and antibiotic withdrawal were quite rapid and could be observed within 24 hours after the change in therapy status. Pyrosequencing of PCR amplified V3/V4 variable regions of 16S rRNA genes showed that representatives of the orders Clostridiales, Lactobacillales, Bacteroidales, Bifidobacteriales, Enterobacteriales, Erysipelotrichales, Coriobacteriales, Desulfovibrionales, Burkholderiales, Campylobacterales and Actinomycetales were detected in the faeces of hens prior to the antibiotic therapy. Tetracycline and streptomycin therapies decreased the prevalence of Bifidobacteriales, Bacteroidales, Clostridiales, Desulfovibrionales, Burkholderiales and Campylobacterales in faecal samples in both experiments. On the other hand, Enterobacteriales and Lactobacillales always increased in prevalence in response to both therapies. Within the latter two orders, Escherichia and Enterococcus were the genera prevalence of which increased after all the antibiotic treatments. CONCLUSIONS: The changes in microbiota composition induced by the antibiotic therapy were rapid and quite dramatic and only representatives of the genera Enterococcus and Escherichia increased in response to the therapy with both antibiotics in both experiments

Virulence potential of five major pathogenicity islands (SPI-1 to SPI-5) of Salmonella enterica serovar Enteritidis for chickens

<p>Abstract</p> <p>Background</p> <p><it>Salmonella </it>is a highly successful parasite of reptiles, birds and mammals. Its ability to infect and colonise such a broad range of hosts coincided with the introduction of new genetic determinants, among them 5 major pathogenicity islands (SPI1-5), into the <it>Salmonella </it>genome. However, only limited information is available on how each of these pathogenicity islands influences the ability of <it>Salmonella </it>to infect chickens. In this study, we therefore constructed <it>Salmonella </it>Enteritidis mutants with each SPI deleted separately, with single individual SPIs (i.e. with the remaining four deleted) and a mutant with all 5 SPIs deleted, and assessed their virulence in one-day-old chickens, together with the innate immune response of this host.</p> <p>Results</p> <p>The mutant lacking all 5 major SPIs was still capable of colonising the caecum while colonisation of the liver and spleen was dependent on the presence of both SPI-1 and SPI-2. In contrast, the absence of SPI-3, SPI-4 or SPI-5 individually did not influence virulence of <it>S</it>. Enteritidis for chickens, but collectively they contributed to the colonisation of the spleen. Proinflammatory signalling and heterophil infiltration was dependent on intact SPI-1 only and not on other SPIs.</p> <p>Conclusions</p> <p>SPI-1 and SPI-2 are the two most important pathogenicity islands of <it>Salmonella </it>Enteritidis required for the colonisation of systemic sites in chickens.</p

Electron microscopy of flagella in <i>S</i>. Enteritidis.

<p>Flagella could be visualised in all the strains and mutants with intact <i>fliC</i> after negative staining with ammonium molybdate.</p

Protective capacity of the SPI1-<i>lon</i>, SPI1-<i>lon</i>-<i>fliC</i> and SPI1-<i>lon</i>-<i>fliC</i>-<i>rcsA</i> mutants after oral-oral vaccination and oral or intravenous challenge in chickens.

#<p>number of positive chickens<b>/</b>number of tested.</p>*<p>t-test different from the non-vaccinated control chickens at P<0.05.</p>&<p>χ² test different from the chickens vaccinated with the SPI1-<i>lon</i>-<i>fliC</i> mutant in caecum at 4 DPI (P<0.05).</p

Persistence, attenuation and protective capacity of the SPI1 and <i>lon</i> mutants for chickens.

&<p>data for days 21 and 42 of life indicate persistence of the vaccine strains, data for days 46 and 56 of life indicate colonization by the challenge wild-type <i>S</i>. Enteritidis.</p>#<p>number of positive chickens<b>/</b>number of tested.</p>∧<p>n.d., not determined due to the small size of some of the spleens of 21-day-old chickens.</p>*<p>significantly different from non-vaccinated controls by χ² test at P<0.05.</p

Protective capacity of the SPI1-<i>lon</i> and SPI1-<i>lon</i>-<i>fliC</i> mutants after oral-oral-intravenous vaccination, followed by oral or intravenous challenge in chickens.

$<p>DPV, days post intravenous vaccination.</p>#<p>number of positive chickens<b>/</b>number of tested.</p>*<p>χ² test different from the non-vaccinated control chickens at P<0.05.</p>&<p>t-test different from the non-vaccinated control chickens at P<0.05.</p

Colony morphology of the wild-type <i>S</i>. Enteritidis, SPI1- <i>lon</i>::Cm-<i>fliC</i> mutant and SPI1-<i>lon</i>::Cm-<i>fliC</i>-<i>rcsB</i>::Kan mutant.

<p>Inactivation of <i>lon</i> resulted in a mucoid colony phenotype which was observed in all the mutants with the <i>lon</i> mutation except for the mutant in which the <i>rcsB</i> mutation has been introduced. The overproduction of capsular polysaccharides in the vaccine strain enables simple differentiation of the vaccine strain from those circulating in the environment.</p

List of primers used in this study for the construction of <i>fliC</i>, <i>rcsB</i> and <i>lon</i> mutants.

*<p>For primers used for the amplification of pKD3 or pKD4, only the gene specific overhangs are shown. „CTR“ primers, either Forward (F) or Reverse (R) were used for the verification of the final contructs.</p