Comparison of two Salmonella enteritidis phage typing schemes

The comparison of two phage typing schemes for Salmonella enteritidis was performed. A total of 517 strains were phage-typed according to the schemes of Lalko [27] and Ward et al. [21]. Strains were isolated from food-poisoning outbreaks and other common sources in Poland, between 1986–1995. Above 99% of all strains tested were differentiated to the definitive phage type using the Lalko collection of typing phages. Phage types 1 and 7 (PTs 1, 7) were the most isolated. The typing phages of Ward enabled to assign 56.5% of all strains (a total of 14 phage types were presented), 37.1% – reacted with phages without showing any of the designated phage types and 6.4% were untypable. Phage type 8 (PT8) predominated. The majority of Salmonella enteritidis strains from one phage type outbreaks of Lalko presented different types of lytic reactions with the Ward phages. Only the correspondence of Salmonella enteritidis PT7 of Lalko with PT8 of Ward et al. [21] was observed.     

Subdivision of Salmonella enteritidis phage types by plasmid profile typing

Differentiation of Salmonella enteritidis by plasmid profile typing has been compared to differentiation by phage typing. Examination of the type strains of the 27 S. enteritidis phage types showed that only 11 profile patterns could be identified. Moreover, two profile patterns were found in 15 of the type strains, including those of the two most common phage types in Britain, types 4 and 8. On this basis, plasmid profile typing is not as sensitive as phage typing for the primary subdivision of S. enteritidis. When differentiation of 534 strains of the 27 phage types was attempted using plasmid profiles, variation in pattern suitable for epidemiological subdivision was found in 13 phage types and there were 9 profile patterns in strains of phage type 4. Plasmid profile typing can, therefore, be regarded as an effective adjunct to phage typing for the subdivision of S. enteritidis.     

Use of plasmid profile typing for surveillance of Salmonella enteritidis phage type 4 from humans, poultry and eggs.

Plasmids were found in 1022 of 1089 (94%) of drug-sensitive strains of Salmonella enteritidis phage type 4 from humans (sporadic and outbreak cases), poultry (chickens) and eggs in England and Wales in the 5-year period 1988-92 and 25 plasmid profile patterns were identified. Strains characterized by a single plasmid of 38 MDa predominated (= plasmid profile type SE 38), comprising over 90% of isolates from humans, 70% from poultry and 92% from eggs. Eleven profile types were identified in strains from humans, 21 in strains from poultry and 3 in strains from eggs. Eight of the 11 patterns identified in human isolates were found in strains from poultry and 2 in strains from eggs. In contrast 15 patterns seen in poultry were not found in strains from humans. Four percent of strains from humans and 13% from poultry did not carry the 38 MDa plasmid but all strains from eggs were found to carry this plasmid. The second most common profile type in strains isolated between 1981 and 1988 was not identified in strains isolated from 1988-92. It is concluded that plasmid profile typing is a useful method for rapid differentiation within phage type 4 of S. enteritidis but that methods which can discriminate within the predominant profile type, SE 38, are now required.     

肠炎沙门菌随机扩增多态性DNA分子分型

目的对不同来源的12株肠炎沙门菌分离株进行分子分型。方法分别提取不同菌株基因组DNA,建立和优化随机扩增多态性DNA（RAPD）反应体系,试用22条随机引物对不同菌株进行RAPD扩增,筛选清晰稳定的扩增条带做统计分析。结果筛选到OPG-03、OPG-06、OPG-07和OPG-13共4条随机引物具有鉴别作用,每一菌株均可扩增出4~10条DNA片段,大多数片段在100~2000bp之间,共扩增出42条RAPD条带,其中共有条带7条,多态性条带35条,多态性为83.3%。不同来源的肠炎沙门菌分离株之间的遗传相似系数在42.6%~100%之间,在0.850的相似性水平上可将12株肠炎沙门菌分离株分为5个不同的亚型。结论应用RAPD技术在分子水平上对肠炎沙门菌进行鉴别和分型具有简便、快速和辨别能力高的优势,对肠炎沙门菌的分子流行病学研究具有较好的应用前景。     

Outer membrane characteristics of Salmonella enteritidis phage type 4 growing in chickens.

Strains of Salmonella enteritidis belonging to phage type 4 (SE4) were grown in the peritoneal cavities of chickens, and without subculture on laboratory media examined for inducible in vivo phenotypic characteristics. These bacteria expressed three major outer membrane proteins (OMPs) of 33, 35 and 36 kilodaltons (kDa), and iron regulated OMPs of 74, 78 and 81 kDa. Bacteria growing in vivo did not express flagella, or fimbriae with a subunit molecular mass of 14 kDa (14 kDa fimbriae). Two OMPs of 55 and 23 kDa, expressed during culture in nutrient broth, were repressed during growth in chickens. Possession of a 38 MDa ''mouse virulence'' plasmid did not influence the expression of OMPs, flagella or fimbriae. It was concluded that strains of SE4 growing in chicken tissues, use an enterobactin mediated iron uptake system to obtain ferric ions, do not express flagella or 14 kDa fimbriae and appear not to express novel OMPs involved in survival in vivo.     

Acquisition of a drug resistance plasmid converts Salmonella enteritidis phage type 4 to phage type 24

Salmonella enteritidis accounted for 55% of the 27,478 salmonellae isolated from humans in England and Wales during 1988. Within this serotype phage type 24 has increased from 24 isolations in 1987 to 201 in 1988. The high frequency of drug resistance in this phage type has been shown to be due to the presence of plasmids belonging to Inc N and coding for resistance to a range of antimicrobial drugs among which resistance to ampicillin, streptomycin, tetracycline (AST) and T predominate. These plasmids are phage-type determining and convert strains of phage type 4 to phage type 24.     

Vaccination of chickens with chicken-derived Salmonella enteritidis phage type 4 aroA live oral Salmonella vaccines.

Two strains of Salmonella enteritidis phage type 4 isolated from chickens, SeLA5 and Se267, were virulent in Balb/c mice by intraperitoneal (i.p.) infection. In 18-20-day-old chickens given 10(7) c.f.u. intravenously (i.v.), both strains caused some deaths. One-day-old chicks infected orally with 10(9) c.f.u. of either strain also suffered limited mortality. Strain SeLA5 was the more virulent by the oral route. AroA mutants SeLA5 aroA and Se267 aroA were reduced in i.p. virulence by up to six logs in the Balb/c mouse model, did not kill 18-20-day-old chickens when injected i.v. and did not multiply in the tissues. Oral vaccination of chicks with either 10(9) c.f.u. at one-day-old or 10(5) c.f.u. at seven-day intervals until day 21 with vaccine strains SeLA5 aroA or Se267 aroA conferred significant protection against intravenous challenge with virulent strain Se109 NalR and reduced intestinal shedding following oral challenge. Seroconversion following oral vaccination was not seen with the lower dose regimen and was low with the higher dose. Seroconversion following oral challenge was marked in unvaccinated birds but much lower in vaccinated birds, suggesting that oral vaccination had prevented invasion by the challenge strain.     

Interrelationships between strains of Salmonella enteritidis.

In contrast to S. typhimurium [19], S. enteritidis is a serotype which has its primary food-animal reservoir in poultry. To date, phage typing has been of paramount importance in studying the epidemiology of this serotype and in particular, has demonstrated the involvement of both poultry meat and whole shell eggs in the transmission of S. enteritidis PT4 to humans. The findings discussed above describe various aspects of the serotype, particularly in relation to the involvement of both LPS and plasmids in its virulence and phage type identity (Fig. 2). These findings have led to an increased understanding of the biology of this serotype, which is of major importance in human food-poisoning in England and Wales at the present time.     

Antimicrobial susceptibility and phage types of Salmonella enteritidis strains isolated in the Lublin area

The study aimed at specifying the value of two traditional methods of typing, using the collection of 241 strains of Salmonella Enteritidis isolated from people and animals in the Lublin area in 1994-1995 from the occasional cases of infections. There were 8 phage types identified among the examined strains. Phage types PT 6 (40.24% of strains) and PT 7 (29.46%) were the most numerous ones. Salmonella Enteritidis was numbered among 38 profiles on the basis of the analysis of resistance to 15 antimicrobial agents. It was found that nitrofurantoin had the lowest efficacy in vitro in relation to the examined collection of Salmonella isolates. High percentage of strains were characterized by lack of susceptibility to streptomycin, neomycin, cefixime, tetracycline and canamycine. While analyzing the profiles of resistance to chemiotherapeutics and the expression of phage type of the examined strains it was observed that only 14.1% of all strains showing resistance to nitrofurantoin represented at the same time phage type PT 6, whereas 10.8% of strains belonging to the same resistance profile indicated PT 7 phage type. In correlated analysis of resistance to chemiotherapeutics and phage type expression of the examined collection of 241 isolates of Salmonella Enteritidis, 18.3% of strains showed distinct and unique set of these two features, which can be used in epidemiological investigations for the preliminary characterization of the bacterial population.     

A large outbreak of Salmonella enteritidis phage type 4 associated with eggs from overseas.

In February 1989 the largest reported outbreak to date in the United Kingdom of Salmonella enteritidis phage type 4 (PT4) infection occurred following a wedding reception at a hotel. One hundred and seventy-three people met the case definition of illness of whom 118 had the organism isolated from their stools. A further 17 were found to be S. enteritidis PT4 positive, but were asymptomatic. Lightly-cooked, egg-based sauces were the epidemiologically proven vehicles of infection. Investigations showed this outbreak to be the first to implicate imported European eggs as the source of infection. An unusual feature of this outbreak was a reported incubation period of less than 3 h for some of the confirmed cases of salmonellosis.     

Molecular characterization of plasmids in Salmonella enteritidis phage types.

Plasmids in selected type strains of 26 of the Salmonella enteritidis phage types have been characterized by restriction enzyme fingerprinting and by DNA-DNA hybridization with oligonucleotide probes for Salmonella plasmid virulence (Spv) genes. With one exception, the fingerprints of the 38 MDa plasmids studied were homogeneous but there was heterogeneity in the fingerprints of 59 MDa plasmids found in 4 of the type strains. However all 38 MDa and 59 MDa plasmids were related as was a 45 MDa plasmid identified in the type strain of phage type 19. A 3.5 kb fragment homologous to SpvC was conserved in Hind III digests of all 38 MDa and 59 MDa plasmids, and in the related 45 MDa plasmid. In contrast a 65 MDa plasmid found in the type strain of phage type 10 was not related to these three plasmid molecular weight groups and did not carry the SpvC gene.     

Four linked outbreaks of Salmonella enteritidis phage type 4 infection--the continuing egg threat

Four outbreaks of Salmonella enteritidis phage type (PT) 4 occurred among guests at functions for which a single commercial caterer supplied food. Retrospective cohort studies were used to describe the epidemiology of three of these outbreaks and identify the vehicle(s) responsible. Of 172 guests at these three events, 47 fitted the clinical case definition for illness and 24 cases were confirmed to have S. enteritidis PT4 infection. Food containing raw egg was identified epidemiologically as the likely vehicle of infection in two of the three outbreaks (odds ratios (OR) and 95% confidence intervals 9.1 (2.2-39.9) and 6.9 (1.2-46.4)). Logistic regression analysis yielded OR = 10.7 (p = 0.0022) and OR = 9.3 (p = 0.015) for egg consumption in two of the outbreaks. These outbreaks highlighted the continuing need to remind the public and commercial caterers of the potential high risks of contracting salmonella from shell eggs. Education of caterers includes advice to obtain eggs and other products from reputable and identifiable suppliers.     

Molecular epidemiology of Salmonella enteritidis phage type 1b and 6a isolates in Portugal

Salmonella enterica serotype Enteritidis is an important serovar comprising 76 % of Salmonella isolates in Portugal in 2001. For better understand the epidemiology of salmonellosis, a total of 47 isolates of S. Enteritidis phage type (PT) 1b and 6a were analysed by pulsed-field gel electrophoresis (PFGE) and genomic DNA was subjected to macro restriction with XbaI. For PT 1b isolates, only three different patterns were observed, and PT6a showed a total of 10 digestion patterns. Curiously, the main pattern among PT1b isolates seams quite similar to main pattern of PT6a isolates, but when the two patterns were analysed with Bionumerics, we observed that they exhibited some differences. It was concluded that, in 2001, there was one predominant pattern for PT1b and PT6a and, possibly, we were in presence of clonal strains that exists all over the country.     

Two outbreaks of Salmonella enteritidis phage type 4 infection associated with the consumption of fresh shell-egg products

In 1988 there were two outbreaks of infection with Salmonella enteritidis phage type 4 in adjacent local authorities. The first affected 18 of 75 helpers and guests who attended a private function. Investigations revealed that home-made vanilla ice-cream containing uncooked eggs was the vehicle of infection and the causative organism was identified at the premises of the egg producer. The second affected 84 of 422 delegates attending a conference dinner, and 12 of 50 hotel staff at risk. A dessert made with lightly-cooked egg yolk and raw egg white was associated with infection, and the epidemic strain was cultured from the shell of an egg and an environmental sample from the producer's farm. It is of interest that one outbreak involved free-range and one battery-produced eggs, and that in one the vehicle was prepared at home and in the other in commercial premises. In neither incident was any deficiency in standards of egg production or catering practice discovered.     

Salmonella enteritidis phage type 4 from the contents of intact eggs: a study involving naturally infected hens

Two small flocks of egg-laying hens, naturally infected with Salmonella enteritidis, were housed in individual cages so that their eggs could be identified. During a longitudinal study where the contents of 1,119 eggs were examined, 11 were positive for S. enteritidis. One isolate was phage type (PT) 33 the others were PT4. The production of infected eggs was clustered though intermittent. The positive eggs, which were produced by 10 of the 35 hens, were all found to contain fewer than 10 salmonellas. Some birds were also apparently carrying S. hadar PT14 as this organism was isolated from the contents of six cracked eggs.     

Correlation of phage type, biotype and source in strains of Salmonella typhimurium

A series of 2092 cultures of Salmonella typhimurium isolated from human, animal and other sources in 57 countries were differentiated into 204 phage types and 19 primary and 147 full biotypes. Different biotypes belonged to the same phage type and different phage types to the same biotype, so the combination of typing methods differentiated strains more finely than either method alone: 574 different `phage type/biotypes'' were distinguished in 1937 cultures belonging to the 204 recognized phage types.