Involvement of MicroRNAs in Probiotics-Induced Reduction of the Cecal Inflammation by Salmonella Typhimurium

The microRNAs (miRNAs) have been shown to play important roles in the development of the immune system and in regulation of host inflammation responses. Probiotics can effectively alleviate the inflammation caused by Salmonella in chickens. However, whether and how miRNAs are involved in modulation of the inflammation response in the gut of chickens have not been reported. In this study, the impact of a probiotics, Lactobacillus plantarum Z01 (LPZ01), was investigated on the cecal miRNAs and cytokine secretions in Salmonella Typhimurium (S. Typhimurium)-infected chickens at the age of 3 days. Newly hatched chicks were assigned to four groups (1): NC (basal diet) (2): S (basal diet + S. Typhimurium challenged) (3): SP (basal diet + S. Typhimurium challenged + LPZ01) (4): P (basal diet + LPZ01). In comparison with the S group, chicks in the SP group reduced the number of S. Typhimurium and had lower levels of interferon-γ and lipopolysaccharide-induced tumor necrosis factor alpha factor (LITAF) in ceca post challenge. Expression of 14 miRNAs was significantly affected by the presence of S. Typhimurium and/or lactobacillus. Five differential expression miRNAs (gga-miR-215-5p, gga-miR-3525, gga-miR-193a-5p, gga-miR-122-5p, and gga-miR-375) were randomly selected for confirmation by the RT-PCR. Predicted target genes of differentially expressed miRNAs were enriched in regulation of cAMP-dependent protein kinase activity, stress-activated MAPK cascade, immune system development and regulation of immune system process as well as in immune related pathways such as MAPK and Wnt signaling pathways. The relationship between changes of miRNAs and changes of cytokines was explored. Finally, 119 novel miRNAs were identified in 36 libraries totally. Identification of novel miRNAs significantly expanded the repertoire of chicken miRNAs and provided the basis for understanding the function of miRNAs in the host. Our results suggest that the probiotics reduce the inflammation of the S. Typhimurium infection in neonatal broiler chicks, at least partially, through regulation of miRNAs expression

The LiaFSR and BsrXRS Systems Contribute to Bile Salt Resistance in Enterococcus faecium Isolates

Two-component systems (TCSs) are dominant regulating components in bacteria for responding to environmental stimuli. However, little information is available on how TCSs in Enterococcus faecium respond to bile salts – an important environmental stimulus for intestinal bacteria. In this study, the gene expression of 2 TCSs, BsrXRS and LiaFSR, was positively correlated with survival rates of different E. faecium isolates during exposure to ox gall. Moreover, gene disruptions of bsrR, bsrS, liaS, and liaR significantly reduced the survival rates of E. faecium in the presence of ox gall. Finally, EMSA results indicated that BsrR functioned as a transcription regulator for expression of its own gene as well as lipoate-protein ligase A (lplA). Additional 27 potential target genes by BsrR were revealed through in silico analyses. These findings suggest that BsrXRS and LiaFSR systems play important roles in bile salt resistance in E. faecium

Using In Vitro Immunomodulatory Properties of Lactic Acid Bacteria for Selection of Probiotics against Salmonella Infection in Broiler Chicks.

Poultry is known to be a major reservoir of Salmonella. The use of lactic acid bacteria has become one of successful strategies to control Salmonella in poultry. The purpose of this study was to select lactic acid bacteria strains by their in vitro immunomodulatory properties for potential use as probiotics against Salmonella infection in broiler chicks. Among 101 isolated lactic acid bacteria strains, 13 strains effectively survived under acidic (pH 2.5) and bile salt (ranging from 0.1% to 1.0%) conditions, effectively inhibited growth of 6 pathogens, and adhered to Caco-2 cells. However, their in vitro immunomodulatory activities differed significantly. Finally, three strains with higher in vitro immunomodulatory properties (Lactobacillus plantarum PZ01, Lactobacillus salivarius JM32 and Pediococcus acidilactici JH231) and three strains with lower in vitro immunomodulatory activities (Enterococcus faecium JS11, Lactobacillus salivarius JK22 and Lactobacillus salivarius JM2A1) were compared for their inhibitory effects on Salmonella adhesion and invasion to Caco-2 cells in vitro and their antimicrobial effects in vivo. The former three strains inhibited Salmonella adhesion and invasion to Caco-2 cells in vitro, reduced the number of Salmonella in intestinal content, spleen and liver, reduced the levels of lipopolysaccharide-induced TNF-α factor (LITAF), IL-1β, IL-6 and IL-12 in serum and increased the level of IL-10 in serum during a challenge study in vivo more efficiently than the latter three strains. These results suggest that in vitro immunomodulatory activities could be used as additional parameters to select more effective probiotics as feed supplements for poultry

Characterization of methicillin-resistant and -susceptible staphylococcal isolates from bovine milk in northwestern china.

Emergence of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MR-CoNS) in bovine milk is a major public health concern. The primary purpose of this research was to determine molecular genetic characteristics and antibiotic resistance of staphylococcal isolates recovered from milk of mastitic cows in the Shaanxi Province in Northwestern China. One hundred and thirteen methicillin-susceptible Staphylococcus aureus (MSSA), one mecA-positive and phenotype-positive MRSA, seven mecA- and mecC- negative but phenotype-positive MRSA and two MR-CoNS including one oxacillin-susceptible mecA-positive Staphylococcus haemolyticus (OS-MRSH) and one mecA-positive and methicillin-resistant Staphylococcus epidermidis (MRSE) isolates were recovered from 214 quarter milk samples on 4 dairy farms. All above 123 isolates were subjected to antibiotic resistance profiling. S. aureus isolates were also genotyped using the spa typing and the multilocus sequence typing (MLST). Eight MRSA and 2 MR-CoNS isolates were additionally tested for SCCmec types. Resistance was common among isolates against ampicillin or penicillin (80.5%), kanamycin (68.3%), gentamicin (67.5%), tetracycline (43.9%) and chloramphenicol (30.1%). However, no isolate was resistant to vancomycin or teicoplanin. Twenty, 29 and 58 isolates showed resistance to 1, 2 or more than 2 antibiotics, respectively. The predominant multidrug resistance profile was penicillin/ampicillin/kanamycin/gentamicin/tetracycline (46 isolates). Most S. aureus isolates belonged to spa types t524 (n = 63), t11772 (a new type, n = 31) and t4207 (n = 15). At the same time, MLST types ST71 (n = 67) and ST2738 (a new type, n = 45) were identified as dominant sequence types. The mecA-positive and phenotype-positive MRSA isolate had a composite genotype t524-ST71-SCCmecIVa, while 7 mecA-negative but phenotype-positive MRSA isolates were all t524-ST71. The OS-MRSH isolate contained a type V SCCmec cassette, while the MRSE isolate possessed a non-typeable SCCmec. The spa-MLST types t11772-ST2738 (n = 27), t11807-ST2683 (n = 4) and t11771-ST2738 (n = 3) were newly identified genotypes of S. aureus. These new genotypes and multidrug-resistant staphylococci could pose additional threat to animal and human health

Cytokine levels in sera of chicks gavaged with LAB strains and followed by <i>Salmonella</i> challenge.

<p>The cytokines (A) LITAF, (B) IL-1β, (C) IL-6, (D) IL-10 and (E) IL-12 were measured by the ELISA methods with specific antibodies of LITAF, IL-1β, IL-6, IL-10 and IL-12 in chicks prepared in our laboratory. Each vertical bar represents the mean ± SD (n = 6). Different letters above bars indicate significant differences among treatments within each sampling day (<i>P</i><0.05).</p

Numbers of <i>S</i>. <i>aureus</i> isolates with different combinations of <i>spa</i> and MLST types.

<p>Numbers of <i>S</i>. <i>aureus</i> isolates with different combinations of <i>spa</i> and MLST types.</p

(A) LITAF, (B) IL-12 production by spleen mononuclear cells after incubation with different LAB strains.

<p>Spleen cells (1×10⁶ cells/ml) were cultured in a RPMI-1640 medium without penicillin or streptomycin. The RPMI-1640 medium alone was used as a negative control, while LPS (lipopolysaccharide, 10 μg/ml) was used as a positive control. LAB (10⁸ to 10⁹ cfu/ml) were cultured with spleen cells. After 24 h and 48 h of culture, LITAF and IL-12 produced in the culture supernatants were analyzed. Each value represents the mean value ± SD from three independent experiments. * indicates significant differences in comparison with the positive control (*: <i>P</i><0.05).</p

Effects of LAB culture on the adhesion and invasion of Caco-2 cells by <i>Salmonella</i> Enteritidis.

<p>The relative adhesion or invasion of <i>S</i>. Enteritidis ATCC13076 to Caco-2 cells was expressed as a percentage using the following formula: relative adhesion or invasion = 100 x A1/A2, where A1 and A2 were the percentages of adhesion or invasion by <i>S</i>. Enteritidis ATCC13076 in the presence and absence of LAB strains, respectively. Each value represents the mean value ± SD from six trials. Different letters above bars indicate significant differences among treatments within each sampling day (<i>P</i><0.05).</p