Enteritidis Isolates Research Articles

Salmonella enteritidis in Western Europe 1995-98 - a surveillance report from Enter-net.

A decline in the incidence of Salmonella enteritidis infections in western Europe was identified by the Salm-Net (forerunner of Enter-net) salmonella database between 1993 and 1995, when the numbers of S. enteritidis isolates fell from 37 647 to 31 482. T

Effect of pH, temperature and surface contact on the elaboration of fimbriae and flagella by Salmonella serotype Enteritidis.

Survival of enteric pathogens exposed to various environmental stresses depends upon a number of protective responses, some of which are associated with induction of virulence determinants. Flagella and fimbriae are putative virulence determinants of Salmonella spp. and ELISAs specific for the detection of flagella and SEF21, SEF14 and SEF17 fimbriae were used to assess the effect of temperature and pH upon their elaboration by isolates of Salmonella serotype Enteritidis in planktonic growth and on the surface of two-dimensional gradient agar plates. For three phage type 4 isolates of Enteritidis of comparative clinical provenance, similar phenotypes for the elaboration of these surface antigens were observed. SEF14 fimbriae were elaborated in planktonic growth at 37 degrees C, but not 20 degrees C, at pH 4.77 and above but not at pH 4.04; whereas on agar gradient plates SEF14 fimbriae were elaborated poorly but with best yields at pH 4.04. SEF17 fimbriae were elaborated in planktonic growth at 20 degrees C, but not at 37 degrees C, at pH 6.18 and above but not at pH 5.09 or below; whereas on agar gradient plates SEF17 fimbriae were elaborated well even at pH 4.65. SEF21 fimbriae were expressed very poorly under all conditions tested. Planktonic growth at 37 degrees C induced least flagella whereas growth at 20 degrees C, and particularly surface growth at lower pH values, induced a 'hyper-flagellate' phenotype. Single colonies allowed to form on gradient agar plates were shown to generate different colonial morphologies which were dependent on initial pH. These results demonstrate that the physicochemical environment is an important determinant of bacterial response, especially the induction of putative virulence factors.

Inter- and intra-phage type differentiation of Salmonella enterica subsp. enterica serovar enteritidis isolates using molecular typing methods

Seventy-six Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) isolates which represented 14 phage types and RDNC isolates were investigated by several independent molecular methods. Eleven different plasmid profiles were detected. Plasmid-encoded virulence genes of the spv gene cluster were shown by hybridization with a spvB/C gene probe to be located on plasmids of either 55 kbp or 95 kbp. Ribotyping as performed with gene probes that recognized either the entire rrn operon or 16S rRNA genes allowed no differentiation between the S. Enteritidis isolates. Three different hybridization patterns could be observed by IS200-typing. Macrorestriction analysis of XbaI-, SpeI- and NotI-digested whole cell DNA proved to be the most discriminatory method as confirmed by the calculation of discriminatory indices. Based on the data obtained from plasmid analysis and macrorestriction analyses with three suitable restriction endonucleases, the 76 S. Enteritidis isolates were assigned to 26 different genomic groups. The observation that several genomic groups included isolates of different phage types has suggested that a combination of suitable molecular methods and phage typing is most useful for tracing S. Enteritidis isolates in epidemiological investigations.

SERE, a widely dispersed bacterial repetitive DNA element.

The presence of a Salmonella serotype Enteritidis repeat element (SERE) located within the upstream regulatory region of the sefABCD operon encoding fimbrial proteins is reported. DNA dot-blot hybridisation analyses and computerised searches of genetic databases indicate that SERE is well conserved and widely distributed throughout the bacterial and archaeal kingdoms. A SERE-based polymerase chain reaction (SERE-PCR) assay was developed to fingerprint 54 isolates of Enteritidis representing nine distinct phage types and 54 isolates of other Salmonella serotypes. SERE-PCR identified five distinct fingerprint profiles among the 54 Enteritidis isolates; no correlation between phage types and SERE-PCR fingerprint patterns was noticed. SERE-PCR was reproducible, rapid and easy to perform. The results of this investigation suggest that the limited heterogeneity of SERE-PCR fingerprint patterns can be utilised to develop serotype- and serogroup-specific fingerprint patterns for isolates of Enteritidis.

Epidemiological analysis of Salmonella enterica Enteritidis isolates in Japan by phage-typing and pulsed-field gel electrophoresis.

Salmonella enterica serotype Enteritidis isolates of phage types (PTs) PT1, PT4, PT13a and PT22 derived from sporadic cases and outbreaks of food poisoning in Japan during 1994 and 1995 were analysed by pulsed-field gel electrophoresis (PFGE). While PT1 strains from 5 different outbreaks showed 14 PFGE patterns, 5 PFGE patterns were observed among PT4 isolates from 5 different outbreaks and 6 independent isolates from imported chicken. Interestingly, 8 out of 9 PT4 strains associated with foreign travel to Southeast Asia were indistinguishable in PFGE pattern from 5 independent isolates of imported chicken from England. Although both PT13a and PT22 were first reported in Japan in 1994, PT22 showed various PFGE patterns compared to PT13a which had the same pattern within an outbreak, unlike PT1. These results could indicate that multiple clonal lines of PT1 and PT22 had already spread while relatively fewer clonal lines of PT4 and PT13a might exist in Japan.

Antibiotic resistance inSalmonella enterica serotype typhimurium

In order to analyse the development of antibiotic resistance in Salmonella spp., a total of 262 Salmonella strains isolated in 1987 (n = 148) and in 1996 (n = 114) from clinical specimens in Wurzburg, Germany, were tested in parallel by the agar diffusion method. In 1987. most of the strains were Salmonella enterica serotype typhimurium (42.6%), whereas in 1996 most were Salmonella enterica serotype enteritidis (68.4%). The majority of Salmonella enterica serotype enteritidis isolates was fully susceptible in 1987 and 1996. In contrast, the percentage of drug-resistant strains of Salmonella enterica serotype typhimurium increased significantly from 27% in 1987 to 52.4% in 1996. This increase, which might reflect uncontrolled use of antibiotics in the environment, should be of concern to public health authorities.

Phage Restriction and the Presence of Small Plasmids in Salmonella enteritidis

Between 1990-1994, a total of 16,505 S. enteritidis strains of human, animal and food origin were phage-typed, using the Hungarian scheme and the changes of incidence of the dominant phage types were monitored. The incidence of PT1 (corresponding to Ward's PT1 was very high between 1990 and 1992 (67.9-71.0% of the total S. enteritidis isolates), later, it decreased. The prevalence of PT6 (corresponding to Ward's PT4) was rare until 1992, then it gradually increased. The phage type and plasmid content of 78 Salmonella enteritidis strains were determined. Small plasmids were present in 59% of the isolates, together with a serotype-specific (38 MDa) plasmid. A correlation was found between the presence of the small plasmid and phage restriction to two phages used for subdividing the Hungarian phage types 1 (PT1) and 6 (PT6) of S. enteritidis (corresponding to PT1 and PT4 in Ward's typing scheme, respectively).

Salmonella bloodstream infection in Ghanaian children.

OBJECTIVE: To examine the frequency of community-acquired salmonella bloodstream infection in Ghanaian children and the occurrence of antibiotic resistance in salmonellae. METHODS: The study comprised 472 patients with a blood culture obtained within 48 h of admission to the pediatric department of Korle Bu Teaching Hospital in Accra, Ghana, over a 3-month period. All Salmonella isolates from blood cultures were speciated and antibiotic susceptibility tests were performed. Clinical data of children with salmonella bloodstream infection were compared to those of controls. Two control groups were identified: all children enrolled in the study without salmonella bloodstream infection (group 1), and those with bloodstream infection due to other organisms (group 2). RESULTS: A pathogen was isolated from 111 children (23.5%), and salmonellae were among the most common isolates (n=24; 21.6%). Among Salmonella strains, S. enteritidis (n=14; 59%) predominated over S. typhi (n=6; 25%). Resistance to several antibiotics was only found in S. enteritidis isolates (n=8; 57%). Children with salmonella bloodstream infection presented more often than controls with severe anemia, jaundice, abdominal pain and distension as well as hepatomegaly and splenomegaly. They were also hospitalized for a significantly longer period, but the case-fatality rate was similar. CONCLUSIONS: Salmonella bloodstream infection, especially due to non-typhoidal strains, is a potential health problem for Ghanaian children and may be complicated by resistance to the commonly available antibiotics.

SEF17 fimbriae are essential for the convoluted colonial morphology of Salmonella enteritidis

Salmonella enteritidis isolated from poultry infections generated a convoluted colonial morphology after 48 h growth on colonisation factor antigen (CFA) agar at 25°C. A mutant S. enteritidis defective for the elaboration of the SEF17 fimbrial antigen, in which the agf gene cluster was inactivated by insertion of an ampicillin resistance gene cassette, and other wild-type S. enteritidis transduced to this genotype failed to produce convoluted colonies. However, growth of SEF17 − mutants at 25°C on CFA agar supplemented with 0.001% Congo red resulted in partial recovery of the phenotype. Immunoelectron microscopy demonstrated that copious amounts of the SEF17 fimbrial antigen were present in the extracellular matrix of convoluted colonies of wild-type virulent S. enteritidis isolates. Bacteria were often hyperflagellated also. Immunoelectron microscopy of SEF17 − mutants grown on CFA agar+0.001% Congo red demonstrated the elaboration of an as yet undefined fimbrial structure. Isolates of S. enteritidis which were described previously as avirulent and sensitive to environmental stress failed to express SEF17 or produce convoluted colonies. These data indicate an essential role for SEF17, and possibly for another fimbria and flagella, in the generation of the convoluted colonial phenotype. The relationship between virulence and colonial phenotype is discussed.

Genotypic Evaluation of Salmonella enteritidis Isolates of Known Phage Types by Arbitrarily Primed Polymerase Chain Reaction

Salmonella enteritidis isolates of known phage types 8, 13a, and 14b were inoculated separately into a group of 14-wk-old specific-pathogen-free chickens. S. enteritidis isolates from pre- and postinoculum were analyzed with the use of arbitrarily primed polymerase chain reaction to determine any genotypic changes after reisolation from the chickens. The preinoculum isolates and the isolates of S. enteritidis recovered at 5, 10, 20, and 35 days postinoculation from the inoculated chickens with various phage types were similar. Therefore, no changes of S. enteritidis organisms of similar phage types occurred at the genotypic level. However, SE-3-B7001 (phage type 8) was changed to phage type 14b after inoculation into the chickens.

Epidemiological analysis of strains of Salmonella enterica serotype Enteritidis from foodborne outbreaks occurring in Italy, 1980-1994.

Polymerase chain reaction (PCR-) ribotyping was performed on 243 strains of Salmonella enterica serotype Enteritidis isolated during the years 1980-1994 from 58 foodborne outbreaks occurring in different regions of Italy. The majority (37) of the outbreaks were attributed to phage type (PT) 4, followed by PT1 (seven outbreaks); the latter was identified in 1993 in Italy in epidemic strains of Enteritidis. In eight cases more than one phage type was recognised from a single event. Nine PCR-ribotypes (PCR-RTs) were detected, with a strong prevalence of PCR-RTs f7 and e5 (23 and 21 outbreaks, respectively). In two instances two distinct PCR-RTs were identified within strains from a single outbreak. All but one of the PT1 outbreaks were caused by PCR-RT f7, whereas PT4 outbreaks could be subdivided into six subsets. Clustering of isolates was consistent with data obtained from epidemiological investigations. PCR-ribotyping proved to be an effective and reliable tool for subtyping isolates of Enteritidis belonging to the most frequent phage types. Nevertheless, in terms of laboratory expertise and lack of inter-laboratory standardisation, this typing technique is best suited for reference laboratories.

Fingerprinting of Salmonella enterica subsp. enterica serovar Enteritidis by ribotyping.

OBJECTIVE: To carry out an epidemiologic evaluation of Salmonella enterica subsp. enterica serovar Enteritidis outbreaks in households and small communities by means of rRNA gene restriction pattern analysis (ribotyping). METHODS: One hundred Enteritidis isolates dating from 1989 to 1994 which could be allocated epidemiologically to different sources or to small community outbreaks were investigated with ribotyping, a fingerprinting method in which bacterial DNA is hybridized with the biotin-labeled plasmid pKK 3535 containing a ribosomal RNA operon of Escherichia coli to determine the ribosomal RNA gene restriction patterns. RESULTS: Four different ribotyping patterns were found with the restriction endonuclease Smal and nine with Sphl. Ribotypes of isolates which could be allocated epidemiologically to a common source usually corresponded. Almost 60% of the Enteritidis infections had the ribotyping pattern Sphl-A. In contrast, this pattern was not found in any of the five Enteritidis strains isolated in 1989. The suspicion that Enteritidis phage type 4 infections are caused by consumption of insufficiently heated eggs is supported by the fact that the ribotyping pattern Sph1-A was found in isolates from eggs and from human specimens. CONCLUSIONS: As patterns Sphl-A and Smal-J appeared in 58% and 75% of the isolates, respectively, ribotyping cannot be used for the differentiation between various outbreaks with these two patterns. In cases where the Enteritidis strains showed less frequent patterns, ribotyping seems to be a practical tool for the identification of infection chains. In addition newly appearing ribotyping patterns can give information about the epidemiologic development of Enteritidis infection.

D values of Salmonella enteritidis isolates and quality attributes of shell eggs and liquid whole eggs treated with irradiation

Irradiation sensitivity of five Salmonella enteritidis isolates inoculated either on the surface or inside of whole shell eggs were determined. The shell eggs were irradiated at doses of 0, 0.5, 1.0, and 1.5 kGy. A minimal dose of 0.5 kGy was sufficient to eliminate all the isolates from the surface of whole eggs; however, the same isolates were more resistant to irradiation when present inside the eggs. The ATCC 13076 isolate was significantly more sensitive to irradiation, with a D value of 0.32 kGy, than the other four isolates from animal origin. Irradiation D values of the latter ranged from 0.39 to 0.41 kGy. Liquid whole eggs were also inoculated (2.4 x 10(6) cells per milliliter) with two S. enteritidis isolates and were heat-treated at 50 C for 0, 20, 40, or 60 min followed by irradiation at 0, 0.25, 0.5, 0.75, or 1.0 kGy. The results indicate that mild heating prior to irradiation was ineffective in reducing the irradiation D values. However, on the basis of the D values obtained, an irradiation dose of 1.5 kGy should be sufficient to reduce Salmonella counts by approximately 4 log10 in both whole shell and liquid eggs. Results also indicate that color and thermal characteristics of the whole or liquid eggs were unaffected by a 1.5-kGy dose of irradiation.

Correlation of conversion of Salmonella enterica serovar enteritidis phage type 1, 4, or 6 to phage type 7 with loss of lipopolysaccharide.

Studies of pairs of Salmonella enterica serovar Enteritidis isolates from different poultry flocks showed that phage type (PT) 7 may be derived from PT 1, 4, and 6. The conversion appeared to be associated with loss of lipopolysaccharide. It is concluded that PT 7 may be of little value as an epidemiological marker of S. enterica serovar Enteritidis.

An epidemiological study of Salmonella enteritidis by pulsed-field gel electrophoresis (PFGE): several PFGE patterns observed in isolates from a food poisoning outbreak.

An epidemiological analysis of Salmonella enteritidis from a food poisoning was done using pulsed-field gel electrophoresis (PFGE) of BlnI- or XbaI-digested fragments of chromosomal DNA of isolates. S. enteritidis isolates obtained from 19 patients had identical PFGE patterns. Therefore, a strain giving the same pattern was considered to be the causative agent of this outbreak. In addition, four isolates that had different BlnI-digested PFGE patterns were obtained from three patients, suggesting that the observed variations in PFGE patterns might occur as the result of some point mutations of chromosomal DNA during growth or from the existence of several S. enteritidis strains from various sources. Subsequent PFGE analysis of continuously subcultured strains supported the former possibility. These observations indicate that PFGE analysis on multiple numbers of colonies from each patient are necessary for the epidemiologic investigation of S. enteritidis.

Intestinal Colonization and Organ Invasion in Chicks Experimentally Infected with Salmonella enteritidis Phage Type 4 and Other Phage Types Isolated from Poultry in the United States

Phage type 4 Salmonella enteritidis strains have only recently been isolated from poultry and humans in the United States, although this phage type predominates in many other countries. The present study assessed the ability of S. enteritidis isolates of various phage types found in the United States (including phage type 4) to colonize the intestinal tract and invade to reach internal organs in experimentally infected chicks. Groups of 5-day-old single-comb white leghorn chicks were inoculated with a range of oral doses of three phage type 4 isolates and three isolates of other phage types. Although some significant differences were observed between individual S. enteritidis isolates in the frequencies at which they colonized the intestinal tracts and invaded to reach the spleens of inoculated chicks, no consistent overall pattern differentiated phage type 4 isolates from isolates of other phage types.

The major sources of Salmonella enteritidis in Thailand

The data of Salmonella serotypes during 1989–1993 from the World Health Organisation (WHO) National Salmonella and Shigella Center, Division of Clinical Pathology, Department of Medical Science, Ministry of Health, Thailand was analysed and found that the prevalence of Salmonella enteritidis had been dramatically increased since 1990. The average S. enteritidis isolates from human patient samples was 0.70% ± 0.41% of the total reported Salmonella isolates during 1972–1989 and increased to 1.33%, 2.98%, 9.54%, and 16.98% in 1990, 1991, 1992, and 1993, respectively. The similar trend of S. enteritidis isolates from chicken meat samples were also observed. However, the conclusive epidemiological relationship between human and chicken S. enteritidis isolates needs to be proved by phage typing or other Salmonella typing methods.

Distribution, gene sequence and expression in vivo of the plasmid encoded fimbrial antigen of Salmonella serotype Enteritidis.

The pefA gene which encoded the serotype associated plasmid (SAP) mediated fimbrial major subunit antigen of Salmonella enterica serotype Typhimurium shared genetic identity with 128 of 706 salmonella isolates as demonstrated by dot (colony) hybridization. Seventy-seven of 113 isolates of Typhimurium and individual isolates of serotypes Bovis-morbificans, Cholerae-suis and Enteritidis phage type 9b hybridized pefA strongly, whereas 48 isolates of Enteritidis hybridized pefA weakly and one Enteritidis isolate of phage type 14b failed to hybridize. Individual isolates of 294 serotypes and 247 individual isolates of serotype Dublin did not hybridize pefA. Southern hybridization of plasmids extracted from Enteritidis demonstrated that the pefA gene probe hybridized strongly an atypical SAP of 80 kb in size harboured by one Enteritidis isolate of phage-type 9b, whereas the typical SAP of 58 kb in size harboured by 48 Enteritidis isolates hybridized weakly. One Enteritidis isolate of phage type 14b which failed to hybridize pefA in dot (colony) hybridization experiments was demonstrated to be plasmid free. A cosmid library of Enteritidis phage type 4 expressed in Escherichia coli K12 was screened by hybridization for the presence of pef sequences. Recombinant clones which were deduced to harbour the entire pef operon elaborated a PEF-like fimbrial structure at the cell surface. The PEF-like fimbrial antigen was purified from one cosmid clone and used in western blot experiments with sera from chickens infected with Enteritidis phage-type 4. Seroconversion to the fimbrial antigen was observed which indicated that the Enteritidis PEF-like fimbrial structure was expressed at some stage during infection. Nucleotide sequence analysis demonstrated that the pefA alleles of Typhimurium and Enteritidis phage-type 4 shared 76% DNA nucleotide and 82% deduced amino acid sequence identity.

RAPID IDENTIFICATION AND SEROTYPING OF SALMONELLA ENTERITIDIS

A latex agglutination kit for S. enteritidis was evaluated on 155 field isolates from 29 serotypes from Denmark, All but one of the S. enteritidis isolates were correctly identified by the kit, and none of the isolates of other serotypes tested were identified as S. enteritidis. The results indicate a very high degree of specificity and sensitivity for the SEF14 monoclonal antibody, which forms the basis of the test.

Treatment of experimental endocarditis due to ampicillin-susceptible or ampicillin-resistant Salmonella enteritidis.

Using two strains of Salmonella enteritidis, one susceptible and one resistant to ampicillin, we studied the efficacies of ampicillin, gentamicin, ampicillin plus gentamicin, ofloxacin, and cefotaxime for the treatment of experimental salmonella endocarditis. Rabbits were treated for 3 days with dosages of antibiotic selected to achieve concentrations in serum equivalent to those obtained in humans during therapy. Aortic salmonella endocarditis seemed to be very difficult to treat, and all antimicrobial regimens failed to achieve the complete sterilization of cardiac vegetations. In vitro studies did not accurately predict the in vivo response to therapy, and no correlations regarding the synergistic activity of the combination of ampicillin plus gentamicin were observed. For the ampicillin-susceptible S. enteritidis isolate, ampicillin and cefotaxime produced the greatest reduction in the number of organisms in vegetations, with no significant differences between them. For the ampicillin-resistant strain, the combination of ampicillin with gentamicin produced a synergistic effect that was not anticipated by the in vitro studies. Both cefotaxime and ofloxacin were effective in reducing the number of microorganisms in the vegetations, although the reduction produced by cefotaxime was less that that produced against the ampicillin-susceptible strain. Monotherapy with gentamicin exhibited only modest activity against the ampicillin-susceptible S. enteritidis strain.