Objective: To analyze the effects of spvD gene on invasion and intracellular proliferation of Caco-2 cells and in order to provide insight into the function of that gene and the underlying mechanism of Salmonella caused infection. Methods: Functional verification of spvD gene deletion mutant and compensation strain. The deletion mutant strain was constructed through a suicide plasmid-mediated homologous recombination. The compensation plasmid constructed by cloning the coding sequence of spvD by PCR into plasmid pBAD33 was mobilized into the deletion mutant by conjugation and the pBAD33 was introduced into wild strains and deleted mutant strains as control. The relative expression of spvD mRNA was detected by quantitative reverse transcription PCR. In order to analyze the virulence of spvD against Caco-2 cells, Caco-2 cells was cocultured with wild type Salmonella enteritidis carrying spvD gene, the deletion mutant strain and compensation strain respectively. The expression level of spvD mRNA and the the number of Salmonella enteritidis after Caco-2 cells intervention were compared between the three groups by LSD-t test, and the invasion rate was compared by χ² test. Results: The expression level of spvD mRNA in wild type Salmonella enteritidis was set as unit "1", the deletion mutant strain was "0.00", and the compensation strain was "2.60" (LSD-t_{wild, deleted}=1.11, P=0.31; LSD-t_{wild, compensation}=-1.77, P=0.13; LSD-t _{deleted, compensation}=-2.88, P=0.03), which confirmed the successful construction of the deletion mutant strain and the compensation strain. The invasion experiment results of the above three Salmonella enteritidis strains on Caco-2 cells showed that the invasion rate of wild strain was 0.23%, the invasion rate of deleted mutant strain was 0.16%, and the invasion rate of compensation strain was 0.16%, with no statistical significance (χ²=1.13, P=0.570). By comparing the number of Salmonella enteritidis at different time points after Caco-2 cells intervention, it was discovered that the number of Salmonella enteritidis in wild strains (6.50×10⁶ CFU/ml) and compensation strains (7.25×10⁶ CFU/ml) was significantly increased than that in deletion mutant strain (1.90×10⁶ CFU/ml) after 16 h coculture (LSD-t_{wild, deleted}=7.95, P=0.00; LSD-t_{wild, compensation}=-1.27, P=0.25; LSD-t _{deleted, compensation}=-9.22, P=0.00). Conclusion: It is not considered that spvD gene can affect the invasion of Salmonella enteritidis on Caco-2 cells, but the gene can promote the reproduction of Salmonella enteritidis in Caco-2 cells.
Caco-2 Cells ; Gene Deletion ; Humans ; Lysergic Acid Diethylamide ; RNA, Messenger/genetics* ; Salmonella enteritidis/genetics*

Salmonella enterica serovar Enteritidis (SE) is one of the most important zoonotic pathogens that cause enteritis and systemic infection in animals and human. Understanding invasive capacities of SE isolates is of vital importance to elucidate pathogenesis of Salmonella infection. To improve the throughput capacity and repeatability of classical gentamicin protection assay (GPA), a modified PGA was developed by taking high-throughput advantage of 96-well cell plates and multichannel pipettes. In addition, drop plate technique rather than spread plate method was applied in the modified GPA protocol for bacterial enumeration. The modified GPA protocol was evaluated by phenotyping intracellular replication of a high virulent and a low virulent SE isolates, JL228 and LN248, in a phagocytic cell line RAW264.7. The protocol was then applied in invasive phenotype determination of 16 SE strains to non-phagocytes (HT-29) and the intracellular replication of 43 SE strains to phagocytes (RAW264.7). Significant lower intra-group and inter-group coefficient of variations of the modified GPA was observed, implying good repeatability and reproducibility over traditional protocol. Further, replication phenotypes were also correlated with those from direct observation by confocal microscopy. Collectively, the improved GPA protocol had advantages of high throughput capacity, good repeatability and reliability, it was also noticed that the protocol also represented a fast and labor-saving alternative scheme for the invasive phenotype determination of Salmonella Enteritidis, and providing reliable phenotype profiles for Salmonella-host interplay interpretation.
Animals ; Gentamicins/pharmacology* ; Humans ; Phenotype ; Reproducibility of Results ; Salmonella Infections, Animal ; Salmonella enteritidis

It is crucial to optimize the dose of fluoroquinolones to avoid antibiotic resistance and to attain clinical success. We undertook this study to optimize the dose of enrofloxacin against Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) in chicken by assessing its pharmacokinetic/pharmacodynamic (PK/PD) indices. The antibacterial activities of enrofloxacin against S. Enteritidis were evaluated. After administering 10 mg/kg body weight (b.w.) of enrofloxacin to broiler chickens of both sexes by intravenous (IV) and peroral (PO) routes, blood samples were drawn at different intervals and enrofloxacin concentrations in plasma were determined. PK/PD indices were calculated by integrating the PK and PD data. The elimination half-lives (T(1/2)), time required to reach peak concentration (T(max)), peak concentration (C(max)), and area under curve (AUC) after administering enrofloxacin by PO and IV routes were 25.84 ± 1.40 h, 0.65 ± 0.12 h, 3.82 ± 0.59 µg/mL, and 20.84 ± 5.0 µg·h/mL, and 12.84 ± 1.4 h, 0.22 ± 0.1 h, 6.74 ± 0.03 µg/mL, and 21.13 ± 0.9 µg.h/mL, respectively. The bioavailability of enrofloxacin was 98.6% ± 8.9% after PO administration. The MICs of enrofloxacin were 0.0625–1 µg/mL against S. Enteritidis strains, and the MIC₅₀ was 0.50 µg/mL. The C(max)/MIC₅₀ were 7.64 ± 0.2 and 13.48 ± 0.7 and the 24 h AUC/MIC₅₀ were 41.68 ± 0.1 and 42.26 ± 0.3 after administering the drug through PO and IV routes, respectively. The data in this study indicate that the application of 50 mg/kg b.w. of enrofloxacin to chicken through PO and IV routes with a dosing interval of 24 h can effectively cure S. Enteritidis infection, indicating the need for a 5-fold increase in the recommended dosage of enrofloxacin in chicken.
Area Under Curve ; Biological Availability ; Body Weight ; Chickens ; Drug Resistance, Microbial ; Fluoroquinolones ; Pharmacokinetics ; Plasma ; Salmonella enterica ; Salmonella enteritidis ; Salmonella ; Serogroup

OBJECTIVES: Gastrointestinal disorders caused by Salmonella enterica serovar Enteritidis (SesE) are a significant health problem around the globe. Probiotic bacteria have been shown to have positive effects on the immune responses. Lactobacillus acidophilus was examined for its capability to influence the innate immune response of HT29 intestinal epithelial cells towards SesE. The purpose of this work was to assess the effect of L. acidophilus PTCC 1643 on cultured intestinal epithelial cells infected with SesE. METHODS: HT29 cells were cultured in Roswell Park Memorial Institute medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were treated with L. acidophilus PTCC 1643 after or before challenge with SesE. At 2 and 4 hours post-infection, we measured changes in the expression levels of TLR2 and TLR4 via real-time polymerase chain reaction. RESULTS: Treatment with L. acidophilus inhibited SesE-induced increases in TLR2 and TLR4 expression in the infected HT29 cells. Moreover, the expression of TLR2 and TLR4 in cells that were pretreated with L. acidophilus and then infected with SesE was significantly higher than that in cells infected with SesE without pretreatment. Taken together, the results indicated that L. acidophilus had an anti-inflammatory effect and modulated the innate immune response to SesE by influencing TLR2 and TLR4 expression. CONCLUSION: Our findings suggested that L. acidophilus PTCC 1643 was able to suppress inflammation caused by SesE infection in HT29 cells and reduce TLR2 and TLR4 expression. Additional in vivo and in vitro studies are required to further elucidate the mechanisms underlying this anti-inflammatory effect.
Bacteria ; Epithelial Cells* ; Gene Expression ; HT29 Cells ; Humans ; Immunity, Innate ; In Vitro Techniques ; Inflammation ; Lactobacillus acidophilus* ; Lactobacillus* ; Probiotics ; Real-Time Polymerase Chain Reaction ; Salmonella enterica* ; Salmonella enteritidis* ; Salmonella*

OBJECTIVES: Few long-term studies have been conducted on the serotype and antibiotic resistance patterns of Salmonella speices (spp.) The aim of this study was to determine the serotypes and antibiotic resistance patterns of Salmonella spp. isolated at Jeollanam-do in Korea from 2004 to 2014. METHODS: A total of 276 Salmonella samples were evaluated. Serotyping was carried out according to the Kauffmann–White scheme. Antibiotic susceptibility was determined using the Vitek II system with an AST-N169 card. RESULTS: A total of 22 different serotypes were identified, and the major serotypes were Salmonella Enteritidis (116 strains, 42.0%) and Salmonella Typhimurium (60 strains, 21.7%). The highest resistance was observed in response to nalidixic acid (43.4%), followed by ampicillin (40.5%) and tetracycline (31.6%). Resistance to nalidixic acid was detected in 81.0% of S. Enteritidis. Multidrug resistance was detected in 43.3% of Salmonella spp. S. Enteritidis and S. Typhimurium presented the highest resistance (98.3%) and multidrug resistance rate (73.3%), respectively. The most highly observed antibiotic resistance pattern among Salmonella spp. in this study was ampicillin-chloramphenicol (14 strains, 5.7%). CONCLUSION: Overall, S. Enteritidis and S. Typhimurium showed higher antibiotic resistance than the other Salmonella serotypes tested in this study. Our study will provide useful information for investigating the sources of Salmonella infections, as well as selecting effective antibiotics for treatment.
Ampicillin ; Anti-Bacterial Agents ; Drug Resistance, Microbial* ; Drug Resistance, Multiple ; Jeollanam-do* ; Korea* ; Nalidixic Acid ; Salmonella enteritidis ; Salmonella Infections ; Salmonella typhimurium ; Salmonella* ; Serogroup* ; Serotyping ; Tetracycline

Lactic acid bacteria (LAB) are commonly used as probiotics in poultry. The present study employed in vitro and in vivo methods to select and test LAB isolated from Muscovy duck ceca as potential probiotics. In the in vitro study, 50 LAB were isolated from Muscovy duck ceca and tested for growth inhibition against Salmonella (S.) Enteritidis. Eleven isolates strongly inhibited S. Enteritidis and only 1 isolate (MD5-2) showing the strongest inhibition was selected for identification. This isolate was called as Lactobacillus (L.) reuteri MD5-2. For the in vivo investigation, 90 1-day-old Muscovy ducks were randomly assigned into three groups of 30 animals each (group 1, control; group 2, treated with 108 colony-forming unit (CFU) of L. reuteri MD5-2 orally once on day 1; and group 3, treated with 108 CFU of L. reuteri MD5-2 orally once daily from days 1 to 5). The ducks were housed in three large cages and raised for 50 days, after which body weight, duodenal villus height and crypt depth were measured. Both villus height and villus height to crypt depth ratio were significantly greater in group 3 than in groups 1 and 2. In conclusion, further investigation of L. reuteri MD5-2 as a potential probiotic strain is warranted.
Animals ; Bacteria ; Body Weight ; Ducks* ; Lactic Acid ; Lactobacillus reuteri* ; Lactobacillus* ; Poultry ; Probiotics* ; Salmonella ; Salmonella enteritidis ; Stem Cells

OBJECTIVETo investigate the antimicrobial resistance status and pulsed field gel electrophoresis (PFGE) patterns of Salmonella Enteritidis (S.Enteritidis) strains in Henan province.

METHODSS. Enteritidis strains were isolated from seven sentinel hospitals from March 2011 to December 2013. According to molecular typing and Salmonella (Kirby-Bauer, K-B) drug susceptibility testing method published by the international PulseNet bacterial infectious disease monitoring network and USA Clinical and Laboratory Standards Institute (CLSI), we analyzed drug sensitivity of 8 kinds antibiotics and PFGE molecule characteristics of 120 S. Enteritidis isolates from seven sentinel hospitals.

RESULTSAmong 120 strains of S. Enteritidis, 77 were isolated from male patients, 43 from female patients. A total of 78 strains S. Enteritidis were isolated from young children ranged from 0 to 5 years old (65.0%), including 57 strains isolated from 6 months to 2 years old (47.5%). The isolated time mainly centralized on May to October of the year, 11 strains isolated in March-April (9.2%), 48 were in May-July (40.0%),54 in August-October (45.0%), 7 in other months, with a typical summer seasonal characteristics. The resistance rate of 120 strains S. Enteritidis to ampicillin was 50.0% (n=60); to ceftazidime was 14.2% (n=17), to cefotaxime was 18.3% (n=22); to cefepime was 5.8% (n=7); to nalidixic acid was 61.7% (n=74); to ciprofloxacin was 8.3% (n=10), to norfloxacin was 5.8% (n=7); to gentamicin was 42.5% (n=51); to streptomycin was 21.7% (n=26); to chloramphenicol was 30.0% (n=36); resistance to methicillin benzyl ammonium was 11.7% (n=14), compound sulfamethoxazole resistance rate was 71.7% (n=86); the tetracycline resistant rate was 47.5% (n=57). All 120 strains of S. Enteritidis had different levels of resistance to 8 kinds of antibiotics, all strains were multidrug resistant strains, 28 isolates were resistant to 3-4 kinds of antibiotics (23.3%), 38 isolates were resistant to 5-6 kinds of antibiotics (31.7%), 39 isolates were resistant to 7-8 kinds of antibiotics (32.5%). All 120 strains of S. Enteritidis were divided into 44 molecular patterns by digestion with XbaI and pulsed field gel electrophoresis. each pattern contained 1-35 strains with similarity ranged from 54.3%-100%. EN14 and EN19 were the main PFGE types, including 35 and 29 strains respectively.

CONCLUSIONThe status of drug resistance of clinical isolates of Salmonella in Henan province was rather serious, PFGE patterns showed advantages and partial strain's corresponding resistant spectrum have certain relevance and the same aggregation relationship.

Anti-Bacterial Agents ; pharmacology ; Child, Preschool ; China ; Drug Resistance, Bacterial ; Electrophoresis, Gel, Pulsed-Field ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Microbial Sensitivity Tests ; Molecular Typing ; Salmonella Infections ; microbiology ; Salmonella enteritidis ; classification ; drug effects

This study was conducted to isolate lactic acid bacteria (LAB) from dog intestine and identify potential probiotic strains for canine use. One hundred and one LAB were isolated from feces of 20 healthy dogs. Acid, bile, and heat resistance along with adherence to Caco-2 cells and antimicrobial activity against pathogens were examined. To analyze immunomodulative effects, the production of nitric oxide (NO), TNF-alpha, and IL-1beta was measured using RAW 264.7 macrophages. Additionally, RAW BLUE cells were used to evaluate nuclear factor-kappaB (NF-kappaB) generation. Ultimately, three strains were selected as canine probiotics and identified as Lactobacillus reuteri L10, Enterococcus faecium S33, and Bifidobacterium longum B3 by 16S rRNA sequence analysis. The L10 and S33 strains showed tolerance to pH 2.5 for 2 h, 1.0% Oxgall for 2 h, and 60degrees C for 5 min. These strains also had strong antimicrobial activity against Escherichia coli KCTC 1682, Salmonella Enteritidis KCCM 12021, Staphylococcus aureus KCTC 1621, and Listeria monocytogenes KCTC 3569. All three strains exerted better immunomodulatory effects than Lactobacillus rhamnosus GG (LGG), a well-known commercial immunomodulatory strain, based on NO, NF-kappaB, IL-1beta, and TNF-alpha production. These results suggested that the three selected strains could serve as canine probiotics.
Animals ; Bacteria* ; Bifidobacterium ; Bile ; Caco-2 Cells ; Dogs ; Enterococcus faecium ; Escherichia coli ; Feces ; Hot Temperature ; Humans ; Hydrogen-Ion Concentration ; Immunomodulation ; Intestines ; Lactic Acid* ; Lactobacillus reuteri ; Lactobacillus rhamnosus ; Listeria monocytogenes ; Macrophages ; NF-kappa B ; Nitric Oxide ; Probiotics* ; Salmonella enteritidis ; Sequence Analysis ; Staphylococcus aureus ; Sulfalene ; Tumor Necrosis Factor-alpha

Disease Outbreaks ; Epidemics ; Foodborne Diseases ; Humans ; Salmonella Food Poisoning ; Salmonella enteritidis

To develop a live vaccine strain against fowl typhoid and paratyphoid caused by Salmonella serovar Gallinarum biovar Gallinarum (Salmonella Gallinarum) and Salmonella serovar Enteritidis (Salmonella Enteritidis), respectively, several nalidixic acid resistant mutants were selected from lipopolysaccharide (LPS) rough strains of Salmonella Gallinarum that escaped from fatal infection of a LPS-binding lytic bacteriophage. A non-virulent and immunogenic vaccine strain of Salmonella Gallinarum, SR2-N6, was established through in vivo pathogenicity and protection efficacy tests. SR2-N6 was highly protective against Salmonella Gallinarum and Salmonella Enteritidis and safer than Salmonella Gallinarum vaccine strain SG 9R in the condition of protein-energy malnutrition. Thus, SR2-N6 may be a safe and efficacious vaccine strain to prevent both fowl typhoid and paratyphoid.
Bacteriophages ; Nalidixic Acid ; Protein-Energy Malnutrition ; Salmonella ; Salmonella enteritidis ; Typhoid Fever* ; United Nations ; Virulence