A clinical isolate of Salmonella enterica serotype Enteritidis in Korea was found to produce the extended-spectrum beta-lactamase CTX-M-15. The isolate was recovered in 2008 from the stool of a 3-yr-old boy with gastroenteritis. This isolate was found to be resistant to multiple drugs, including ampicillin, piperacillin, cefotaxime, ceftazidime, cefepime, and aztreonam. The resistance to cefotaxime was transferred by conjugation to recipient Escherichia coli J53. The patient was eventually successfully treated with trimethoprim-sulfamethoxazole. This is the first report of the bla (CTX-M-15) gene in S. enterica serotype Enteritidis in Korea.
Child, Preschool ; Gastroenteritis/diagnosis/*microbiology ; Humans ; Male ; Salmonella enteritidis/genetics/*isolation & purification ; Serotyping ; beta-Lactamases/*genetics

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*

OBJECTIVETo compare the discriminatory ability between multilocus sequence typing system (MLST) and pulsed-field gel electrophoresis (PFGE).

METHODSSalmonella enteritidis strains, isolated from food in China were identified by MLST, under PCR products of thrA, purE, sucA, aroC, hemD, dnaN and Sdf I. The same set of strains was typed by PFGE using Xba I, Spe I as the restriction enzyme in order to compare the discriminatory power of the methods.

RESULTSData from MLST revealed the lack of diversity among the strains of the same serotype and the number of variable nucleotide sites per locus ranged from 1 to 23 between Salmonella typhi LT2 and other serotypes of Salmonella. However, 50 Salmonella enteritidis strains were identified as 11 patterns and more sub-patterns by PFGE.

CONCLUSIONIn strain typing, PFGE was the highly discriminatory method comparing to the MLST system.

Electrophoresis, Gel, Pulsed-Field ; Genes, Bacterial ; Polymerase Chain Reaction ; Salmonella enteritidis ; classification ; genetics ; Sequence Analysis, DNA ; Serotyping

OBJECTIVETo evaluate the feasibility of the application of variable-number tandem repeat (VNTR) loci of Salmonella Enteritidis (S. enteritidis) in subtyping mutiple-locus variable-number tandem repeat analysis (MLVA).

METHODSA total of 16 isolates of S.enteritidis from different place and time in China were preliminarily assessed by choosing 11 reported VNTR loci, the loci with single amplified bands were picked to subtype all 104 S. enteritidis isolates. The isolates were also analyzed by pulse field gel electrophoresis (PFGE) to compare the superiority or inferiority of MLVA method and PFGE method.

RESULTSSeven VNTR loci were selected from the preliminary screening to expand the analysis, and the 7 VNTR loci had grouped 104 of S.enteritidis isolates into either 16 MLVA subtypes or 22 PFGE subtypes, with the D value at 0.7222 and 0.7974, respectively. Comparing with the isolates in MLVA subtypes, the isolates in PFGE showed a stronger resolving power. Meanwhile the results in PFGE showed a more disperse frequency distribution than those in MLVA.

CONCLUSIONThese results indicate that some VNTR locus which have shown a good polymorphism internationally, may fail to show polymorphism in China, thereby, more VNTR loci should be included in MLVA and the wide screening may benefit the unity of global laboratorial methods.

Bacterial Typing Techniques ; methods ; Electrophoresis, Gel, Pulsed-Field ; Minisatellite Repeats ; Multilocus Sequence Typing ; methods ; Salmonella enteritidis ; classification ; genetics

The best enrichment broth and DNA extraction scheme was determined for rapid and sensitive detection of Salmonella Enteritidis in steamed pork using real-time PCR. The inhibitory effect of commonly used Salmonella enrichment broths, Rappaport-Vassiliadis (RV) and Muller-Kauffmann tetrathionate with novobiocin (MKTTn), on real-time PCR was confirmed. The inhibition of PCR was statistically significant (p < 0.05) in RV and MKTTn, as compared with buffered peptone water (BPW) or phosphate-buffered saline. The inhibitory effect of the selective enrichment media was successfully removed by using a modified DNA extraction, PrepMan Ultra Reagent with an additional washing step or the DNeasy Tissue Kit. In three experiments, when applied to detection of Salmonella Enteritidis in steamed pork, the real-time PCR coupled with single 24 h enrichment with BPW performed better than double 48 h enrichment with BPW plus RV or MKTTn. The simple real-time PCR assay using BPW proved to be a rapid and sensitive test for detection of low concentrations of Salmonella Enteritidis in steamed pork samples as compared with the conventional culture method.
Animals ; Culture Media ; DNA, Bacterial/chemistry/genetics ; *Food Microbiology ; Humans ; Meat/*microbiology ; Polymerase Chain Reaction/methods ; Salmonella Infections/*prevention & control ; Salmonella enteritidis/genetics/*isolation & purification ; Specimen Handling/methods ; Swine

Antibiotic resistance in Salmonella enteritidis and S. typhimurium, one of most frequent etiologic pathogens of food-borne bacterial gastroenteritidis in humans, is a serious health problem worldwide. Fifteen and 22 each of S. enteritidis and S. typhimurium were isolated from animals from 1983 to 1999 in Korea and tested for their antibiotic resistance patterns and phage types. S. enteritides isolates were highly resistant to sulfonamides (86.7%) and four of them (26.6%) showed multiple antibiotic resistance. The most frequent phage type (PT) of S. enteritids was PT1 (33.3%) even though none of them had multiple antibiotic resistance. S. typhimurium isolates were highly resistant to streptomycin, sulfonamides, and tetracycline, 100%, 95.5%, and 86.4% respectively. The incidence of multiple antibiotic resistance of S. typhimurium isolates was extremely high (100%) comparing to S. enteritidis isolates (26.7%). Two of the five ACSSuT type S. typhimurium isolates, resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline, were phage type DT104. All S. typhimurium isolates were sensitive to florfenicol. For the rapid detection of multiple antibiotic resistant S. enteritidis and S. typhimurium isolates, particularly ACSSuT type S. typhimurium DT104, antibiotic resistance genes, cmlA/tetR, PSE-1, and TEM, and Salmonella spp. Specific gene, SipB/C, were amplified using four pairs of primers in hot-started multiplex polymerase chain reaction. Two Korean isolates of S. typhimurium DT104 showed TEM amplicons instead of PSE-1 for the ampicillin resistance. The multiplex PCR used in this study was useful in rapid detection of ACSSuT type S. typhimurium and identification of b-lactamase gene distribution among Salmonella isolates.
Animals ; Anti-Bacterial Agents/*pharmacology ; Bacteriophage Typing ; Base Sequence ; Drug Resistance, Bacterial/genetics ; Drug Resistance, Multiple, Bacterial/genetics ; Gene Amplification ; Humans ; Microbial Sensitivity Tests/veterinary ; Phenotype ; Polymerase Chain Reaction/veterinary ; Salmonella Infections, Animal/drug therapy/*microbiology ; Salmonella enteritidis/classification/*drug effects/genetics ; Salmonella typhimurium/classification/*drug effects/genetics

Protein identification by peptide mass fingerprinting using matrix-assisted laser desorption ionization time of fight (MALDI-TOF) mass spectrometry (MS) can analyze unambiguously identity of the spots from a 2-dimensional electrophoresis (2-DE) gel. This study developed a technique for 2-DE of Salmonella enterica serovar Enteritidis (S. enteritidis) by improving the dissolution conditions by 2-DE using a pH 4 - 7 immobilized pH gradient (IPG) strip. This report examines the protein components from the patterns of the S. enteritidis protein. The most abundant protein displayed a great number of clusters within the pH 4.5 - 7 range with a molecular mass ranging from 35-80 kDa. Some of these spots were identified as metabolic related enzymes. The protein fraction was also analyzed using an immobilized pH gradient strip. Different proteins were identified on the spot according to the elongation factors. In addition, this study showed that the 2-DE analysis of S. enteritidis provides useful information regarding the S. enteritidis proteome, and this approach might provide a strategy for identifying bacterial proteins using a proteome technology.
Bacterial Proteins/analysis/*chemistry/isolation & purification ; Electrophoresis, Gel, Two-Dimensional ; Enzymes/chemistry/genetics/isolation & purification ; Molecular Weight ; Salmonella enteritidis/*chemistry/growth & development ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

A total of 22 Salmonella enterica serotype Enteritidis (S. Enteritidis) strains isolated from human and chicken were subjected to DNA fingerprinting by repetitive sequence PCR using ERIC and BOX primers, antibiotic resistance and plasmid patterns. Both ERIC and BOX PCR amplification data revealed a highly genetic homogeneity between isolates from human and chicken except one isolate, which originated from chicken and showed a different DNA band pattern from others. Eleven of 22 S. Enteritidis isolates (50%) were resistant to more than one antibiotics and characterized by 5 resistance patterns. The most common pattern was penicillin resistant (63.6%). Only one isolate from chicken showed a multiple drug resistance patterns to 4 antibiotics. All 22 S. Enteritidis isolates harbored more than two plasmids with eight different plasmid profiles including two to six plasmids with approximate molecular size ranging from 1.9 to 21 kb. A band of 15 kb size was detected in all isolates tested, however, the band sizes smaller than 15 kb were found only in isolates from chicken.
Animals ; *Chickens ; China/epidemiology ; DNA Fingerprinting/veterinary ; DNA, Bacterial/chemistry/genetics ; Disease Outbreaks/*veterinary ; Humans ; Microbial Sensitivity Tests/veterinary ; Microsatellite Repeats/genetics ; Plasmids/chemistry/genetics ; Polymerase Chain Reaction/veterinary ; Poultry Diseases/epidemiology/*microbiology ; Salmonella Food Poisoning/epidemiology/*microbiology ; Salmonella enteritidis/drug effects/*genetics/isolation&purification