Relationship of pulsed-field profiles with key phage types of Salmonella enterica serotype Enteritidis in Europe: results of an international multi-centre study.

Salmonella is one of the most common causes of foodborne infection in Europe with Salmonella enterica serovar Enteritidis (S. Enteritidis) being the most commonly identified serovar. The predominant phage type for S. Enteritidis is phage type (PT) 4, although PT 8 has increased in incidence. Within these phage types, pulsed-field gel electrophoresis (PFGE) provides a method of further subdivision. The international project, Salm-gene, was established in 2001 to develop a database of PFGE profiles within nine European countries and to establish criteria for real-time pattern recognition. It uses DNA fingerprints of salmonellas to investigate outbreaks and to evaluate trends and emerging issues of foodborne infection within Europe. The Salm-gene database contains details of about 11 700 S. Enteritidis isolates, demonstrating more than 65 unique PFGE profiles. The clonal nature of S. Enteritidis is evidenced by the high similarity and distribution of PFGE profiles. Over 56% (6603/11 716) of the submitted isolates of several different phage types were profile SENTXB.0001, although this profile is most closely associated with PT 4. The next most common profiles, SENTXB.0002 and SENTXB.0005, were closely associated with PT 8 and PT 21 respectively. Studies to investigate the relationship of profile types with outbreaks and possible vehicles of infection suggest that the incidence of PFGE profile SENTXB.0002, and thus PT 8, in some countries may be due to importation of foods or food production animals from Eastern Europe, where PT 8 is amongst the most frequently identified phage types. Collation of subtyping data, especially in the commonly recognized phage types, is necessary in order to evaluate trends and emerging issues in salmonella infection.

[Surveillance of infectious foodborne diseases after enactment of the German Protection against Infection Act in 2001. Potentials and requirements]. / Surveillance Lebensmittel-übertragener Infektionserkrankungen durch das Infektionsschutzgesetz: Möglichkeiten und Anforderungen.

The epidemiology of infectious foodborne diseases has changed. Outbreaks more frequently occur geographically dispersed or protractedly over longer periods of time, and they often appear as a scatter of seemingly sporadic cases. This hampers and delays the identification of their epidemiological link. The surveillance of infectious foodborne diseases has to be refined accordingly to be able to detect these diffuse outbreaks. The German Protection against Infection Act, enacted in 2001, offers the potential of increased sensitivity due to timely electronic reporting of individual cases and detailed data accompanying each report. In addition to a timely and comprehensive reporting system, subtyping of pathogens has become an invaluable tool in identifying epidemiologically linked cases, i.e. outbreaks. Still, the sensitivity of foodborne disease surveillance still hinges on the willingness of physicians to order stool testing for enteric pathogens (and to report suspected outbreaks to local health departments). Without the active participation of physicians, the chance of detecting outbreaks and successfully investigating them is markedly reduced. Consequently, the general preventive strategy would be jeopardised, namely to understand the (often new) mechanisms by which contamination and disease transmission occur well enough to interrupt them.

Enterobacterial autoinducer of growth enhances shiga toxin production by enterohemorrhagic Escherichia coli.

The addition of the enterobacterial autoinducer of growth to nutrient-poor minimal medium markedly accelerated the exponential growth rates of strains of enterohemorrhagic Escherichia coli but had little or no effect on maximal cell densities in stationary phase. Growth in the presence of the autoinducer resulted in an approximately twofold enhancement in Shiga toxin production.

Distribution of molecular subtypes within Salmonella enterica serotype Enteritidis phage type 4 and S. Typhimurium definitive phage type 104 in nine European countries, 2000-2004: results of an international multi-centre study.

This study investigates the distribution of pulsed-field gel electrophoresis (PFGE) profiles within Salmonella enterica serotype Enteritidis phage type (PT) 4 and S. Typhimurium definitive phage type (DT) 104, from cases of human infection in nine European countries from 2000 to 2004. Isolates were subtyped using standardized methods and gel images submitted by each participating country to the coordinating centre (Health Protection Agency Centre for Infections, London, UK), where they were entered into a central database, developed within BioNumerics software, and designated using an agreed nomenclature. S. Enteritidis PT4 (n=3637) was differentiated into 38 different profiles. Simpson's index of diversity (D) of profiles ranged from 0.2 to 0.4. Profile SENTXB.0001 represented at least 80% of all profiles in each country. S. Typhimurium DT104 (n=1202) was differentiated into 28 different profile types. Simpson's D was at least 0.6 in all countries except in Austria and Italy. In both these countries over 74% of S. Typhimurium DT104 profiles were STYMXB.0013. Profile STYMXB.0061, was predominant in Denmark, Spain, Finland and England and Wales where it represented between 36% and 45% of profiles. Profile STYMXB.0001 represented nearly half of all profiles in Scotland and 23% in England and Wales. PFGE is proving useful for further discrimination within S. Enteritidis PT4 and S. Typhimurium DT104. Ascertainment of international outbreaks involving common serotypes and phage types may be increased by the timely pooling of PFGE profiles within a central database readily accessible to all participating countries.

Catecholate receptor proteins in Salmonella enterica: role in virulence and implications for vaccine development.

Three outer membrane proteins of Salmonella enterica serovar Typhimurium function as catecholate siderophore receptors. IroN promotes uptake of enterobactin, salmochelins and 2,3-dihydroxybenzoylserine, FepA transports enterobactin and 2,3-dihydroxybenzoylserine, and Cir is a receptor for 2,3-dihydroxybenzoylserine. In addition, all three proteins are required for l-norepinephrine-facilitated iron uptake from transferrin as judged by failure of a fepA iroN cir triple mutant to grow in serum-containing medium in the presence of l-norepinephrine. Moreover, pre-treatment of mice with l-norepinephrine resulted in enhanced systemic spread of the parental strain, but had no effect on the fepA iroN cir mutant. Inoculation of mice with the triple mutant, which is significantly attenuated, elicited a significant protective effect against subsequent challenge with the parental strain.

Molecular epidemiology of Salmonella enterica serovar Agona: characterization of a diffuse outbreak caused by aniseed-fennel-caraway infusion.

During 2002-2003 increased numbers of notified salmonellosis due to S. enterica serovar Agona were observed in Germany. In order to understand the recent spread of this serovar and to trace the route of infection to its source, a new phage-typing scheme and pulsed field gel electrophoresis (PFGE) were used to analyse these isolates. By using 14 bacteriophages, 52 phage types were distinguished among the S. Agona strains. PFGE also differentiated 52 different patterns. A combination of both methods generated 94 clonal types among 165 S. Agona strains originating from Germany and other countries including the United States, United Arab Emirates, Turkey, India, Austria and Finland, indicating a great biological diversity within this serovar. However, 36 recent S. Agona isolates from infantile gastroenteritis in Germany, from an untreated batch of aniseed imported from Turkey and from fennel-aniseed-caraway infusion (packed in tea bags) revealed clonal identity indicating their epidemiological relatedness as a new source of infection. It is suggested that strains of S. Agona will continue to be of public health concern, and that phage typing together with PFGE typing should be applied as reliable and rapid tools for epidemiological subtyping and future monitoring.

Pilot study to evaluate microarray hybridization as a tool for Salmonella enterica serovar Typhimurium strain differentiation.

In developed countries, Salmonella enterica subspecies 1 serovars Enteritidis and Typhimurium range among the most common causes of bacterial food-borne infections. The surveillance and typing of epidemic Salmonella strains are important tools in epidemiology. Usually, Salmonella enterica subspecies 1 serovars are differentiated by serotyping for diagnostic purposes. Further differentiation is done by phage typing as well as molecular typing techniques. Here we have designed and evaluated a prototype DNA microarray as a tool for serovar Typhimurium strain differentiation. It harbors 83 serovar Typhimurium probes obtained by differential subtractive hybridization and from the public database. The microarray yielded reproducible hybridization patterns in repeated hybridizations with chromosomal DNA of the same strain and could differentiate five serovar Typhimurium reference strains (DT204, DT104, DT208, DT36, and LT2). Furthermore, the microarray identified two distinct groups among 13 serovar Typhimurium DT104 strains. This correlated with observations from pulsed-field gel electrophoresis analysis. Twenty-three further serovar Typhimurium strains were analyzed to explore future directions for optimization of the simple 83-probe DNA microarray. The data presented here demonstrate that DNA microarrays harboring small numbers of selected probes are promising tools for serovar Typhimurium strain typing.

An outbreak of Salmonella München in Germany associated with raw pork meat.

In summer 2001, an outbreak of Salmonella München occurred in Germany. We conducted descriptive epidemiology and hypothesis-generating interviews among case patients, two retrospective cohort studies, and a case-control study of suboutbreaks. We performed pulsed-field gel electrophoresis (PFGE) from selected patient isolates and a limited trace-back investigation for analytical purposes. Four states were consecutively affected: Saxonia (SX), Brandenburg (BB), Berlin (BE), and Baden-Württemberg (BW). Although hypothesis-generating interviews failed to identify a plausible food item, descriptive data and investigations of the suboutbreaks suggested pork meat as a probable source in three states (SX, BB, and BE) but not in BW. The PFGE profiles from isolates of case patients in the first three states were indistinguishable but differed from PFGE profiles of case patients in BW. Trace-back investigation suggested that contamination of pork meat occurred early in the rearing-production chain. This outbreak demonstrates how contamination early in the production process that can yield different end products may complicate multistate outbreaks. Investigation of suboutbreaks and use of the trace-back method as investigational tools may be useful adjuncts in solving the problem of multistate outbreaks.

Assessment of a new selective chromogenic Bacillus cereus group plating medium and use of enterobacterial autoinducer of growth for cultural identification of Bacillus species.

A new chromogenic Bacillus cereus group plating medium permits differentiation of pathogenic Bacillus species by colony morphology and color. Probiotic B. cereus mutants were distinguished from wild-type strains by their susceptibilities to penicillin G or cefazolin. The enterobacterial autoinducer increased the sensitivity and the speed of enrichment of B. cereus and B. anthracis spores in serum-supplemented minimal salts medium (based on the standard American Petroleum Institute medium) and buffered peptone water.

Presence of sopE gene & its phenotypic expression among different serovars of Salmonella isolated from man & animals.

BACKGROUND & OBJECTIVES: Salmonellae cause a spectrum of diseases in man and animals but their virulence factors responsible for induction of gastroenteritis and/or systematic infection are still poorly understood. Also, the different subspecies and serovars of Salmonella differ considerably in their virulence for man and animals. There is increasing evidence that Salmonella possesses a dedicated protein secretion system denoted type III secretion system (TTSS) that is involved in the early stage of Salmonella infection. One such TTSS is Salmonella outer protein E (SopE) that helps in the invasion of Salmonella by stimulating membrane ruffling. In the present study the presence of sopE gene and its phenotypic expression (SopE protein) among different serovars of Salmonella enterica isolated from man and animals in India was investigated. METHODS: A total of 50 isolates of S. enterica belonging to 11 serovars were tested for the presence of sopE gene by polymerase chain reaction. The in vitro phenotypic expression of SopE protein was detected by Western blotting using anti-SopE serum. RESULTS: Of the 50 isolates of S. enterica belonging to 11 serovars tested for the presence of sopE,14 belonging to three serovars viz., Enteritidis, Gallinarum and Virchow were found to carry the sopE gene. Similarly, 13 isolates belonging to same three serovars were found to express SopE protein phenotypically as detected by Western blotting using anti-SopE serum. INTERPRETATION & CONCLUSION: The results indicated that sopE gene appeared to be distributed and conserved among only a few serovars of Salmonella (Enteritidis, Gallinarum and Virchow) irrespective of their source of isolation. The presence of sopE gene in Salmonella provides an important pathogenic means to invade epithelial cells.

Prevalence and deletion types of the pathogenicity island ETT2 among Escherichia coli strains from oedema disease and colibacillosis in pigs.

Piglet pathogenic Escherichia coli encoding Shigatoxin 2e and F18 adhesins are the etiological agents of oedema disease as well as of non-oedema disease colibacillosis. In order to reveal virulence differences among this pathogen, the presence of the pathogenicity island (PAI) E. coli type three secretion system 2 (ETT2) was examined. Using PCR and Southern blot techniques for the identification of the right, the middle, and the left region of this 29.9kb large genetic element, the entire ETT2 was found among E. coli O138:H(-), O139:H1, and O147:H6 strains originated from cases of oedema disease in Germany between 1995 and 2001 and belonging to various clonal types. In contrast, non-oedema disease E. coli isolates (e.g. O8:H19, 101:H(-), O141:H4) contain deleted subtypes of ETT2. These deletions cover the translocon part of the putative ETT2-encoded type III secretion apparatus. It is suggested that the entire ETT2 is associated with a particular virulence trait of piglet oedema disease E. coli (EDEC).

Trends in antimicrobial drug resistance in Salmonella enterica serotypes Typhi and Paratyphi A isolated in Europe, 1999-2001.

Results of antimicrobial sensitivity tests for strains of Salmonella enterica serotypes Typhi and Paratyphi A isolated from patients in ten European countries between 1999 and 2001 have been transferred electronically to the Enter-net surveillance hub. For Typhi between 22 and 29% of isolates were multiresistant (to four drugs or more) with decreased susceptibility to ciprofloxacin (MIC 0.25-1.0 mg/l) increasing from 20% in 1999 to 26% in 2001. Nineteen of 169 (11%) strains with decreased ciprofloxacin susceptibility were sensitive to nalidixic acid. For Paratyphi A multiple resistance increased from 9% in 1999 to 25% in 2001 and decreased ciprofloxacin susceptibility from 6 to 17%. Clinicians should be aware of the possibility of treatment failures when fluoroquinolones are used as the first-line drug for infections with Typhi and Paratyphi A, particularly for patients recently returning from areas where drug-resistant strains are endemic.

Role of receptor proteins for enterobactin and 2,3-dihydroxybenzoylserine in virulence of Salmonella enterica.

Single, double, and triple mutants of an enterobactin-deficient mutant strain of Salmonella enterica serovar Typhimurium were constructed that were defective in the expression of the iron-regulated outer membrane proteins (IROMPs) FepA, IroN, and Cir, which are proposed to function as catecholate receptors. Uptake of naturally occurring and chemically synthesized catecholate molecules by these mutants was assessed in standard growth promotion assays. Unique patterns of uptake were identified for each IROMP; specifically, FepA and IroN were confirmed to be required for transport of enterobactin, and all three proteins were shown to function as receptors for the enterobactin breakdown product 2,3-dihydroxybenzoylserine. The fepA, iroN, and cir alleles were transduced to enterobactin-proficient strains of S. enterica serovar Typhimurium and S. enterica serovar Enteritidis, and the resulting phenotypes were confirmed by analysis of outer membrane protein profiles, by sensitivity to KP-736, a catecholate-cephalosporin conjugate, and by growth promotion tests on egg white agar. Intragastric infections of mice with the S. enterica serovar Typhimurium strains indicated that the parental strain and the fepA iroN double mutant were similarly virulent but that the fepA iroN cir triple mutant was significantly attenuated. Moreover, in mixed infections, the fepA iroN mutant showed similar cecal colonization and invasion of the liver to the parental strain, while the triple mutant showed significantly reduced cecal colonization and no measurable spread to the liver. Infections of 4-day-old chicks with S. enterica serovar Enteritidis strains also indicated that mutation of the fepA iroN genes did not significantly reduce cecal colonization and systemic spread compared with those of the parental strain. The results indicate that, while enterobactin uptake is not essential for the virulence of S. enterica serovars in mouse and chicken infection models, the ability to take up 2,3-dihydroxybenzoylserine via any of the three catecholate siderophore receptors appears to play an important role, since the S. enterica serovar Typhimurium triple mutant was significantly attenuated in the mouse model. Salmochelins appear not to be involved in the virulence of S. enterica.

Strong association between shiga toxin-producing Escherichia coli O157 and virulence genes stx2 and eae as possible explanation for predominance of serogroup O157 in patients with haemolytic uraemic syndrome.

The aim of this study was to investigate the association between Shiga toxin-producing Escherichia coli (STEC) O157 and the simultaneous presence of the virulence genes stx2 and eae in STEC from patients with gastroenteritis. In Germany, the proportion of serogroup O157 is substantially higher among STEC isolates from patients with haemolytic uraemic syndrome (HUS) than among STEC isolates from patients with gastroenteritis. The reason for this is unknown. Independent of serogroup, the virulence genes stx2 and eae have been associated with severe disease. Data collected in 2000-2001 from a Germany-wide laboratory-based surveillance system for STEC-associated gastroenteritis in patients <15 years were analysed. Overall, 18% of the STEC isolates belonged to serogroup O157. Compared with non-O157 strains, 0157 isolates were strongly associated with the simultaneous presence of both an stx2 gene and the eae gene (OR, 76; 95%CI, 27-230). Within the subset of STEC isolates that carried both virulence genes, 60% belonged to serogroup O157, a proportion similar to that found in STEC isolates from pediatric patients with HUS in Germany and Austria (67%, P=0.35). These data suggest that the more frequent carriage of both virulence genes, i.e. stx2 and eae, forms the basis of why STEC O157 predominates in patients with HUS.

A European outbreak of Salmonella enterica serotype Typhimurium definitive phage type 204b in 2000.

OBJECTIVE: To describe the clinical, epidemiologic and microbiological features of a large outbreak of infection with a multiresistant Salmonella enterica serotype Typhimurium definitive type DT204b infection involving at least 392 people in five European countries. METHODS: Icelandic public-health doctors responded to a report on an Internet news site of an outbreak of infection with a multiresistant strain of Typhimurium DT104 in England by contacting the Public Health Laboratory Service (PHLS) Communicable Disease Surveillance Centre (CDSC). An international alert was sent out through Enter-net. All strains from England & Wales, The Netherlands, Scotland and Germany, and 17 of the outbreak isolates from Iceland, were phage-typed, screened for antimicrobial resistance, and subjected to molecular typing. Hypothesis-generating interviews were conducted, followed by case-control studies performed in Iceland and England. RESULTS: Isolates from cases in Iceland, England and Wales, The Netherlands, Scotland and Germany were identified as Typhimurium DT204b. The antimicrobial resistance pattern was ACGNeKSSuTTmNxCpL. All strains tested displayed an identical plasmid profile. Strains from five cases in England & Wales and five cases in Iceland possessed identical pulsed-field profiles. Although a common source was suspected, only Iceland implicated imported lettuce as a vehicle, with an analytic epidemiologic study (OR = 40.8; P = 0.005; 95% CI 2.7-3175). CONCLUSION: The identification of international outbreaks, necessary for investigation and control, can be facilitated by standardized phage-typing techniques, the electronic transfer of molecular typing patterns, formal and informal links established through international surveillance networks, and the early reporting of national outbreaks to such networks.

Antimicrobial drug resistance in isolates of Salmonella enterica from cases of salmonellosis in humans in Europe in 2000: results of international multi-centre surveillance.

The Enter-net surveillance system received results of antimicrobial sensitivity tests for isolates from over 27 000 cases of human salmonellosis in 2000 in 10 European countries. Almost 40% of isolates were resistant to at least one antimicrobial, with 18% multiresistant. Resistance to ampicillin, streptomycin, sulphonamides and tetracyclines was common, with over 20% of isolates resistant to at least one of these antimicrobials. Clinical resistance to ciprofloxacin was rare, with only 0.5% of isolates exhibiting such resistance (MIC >1.0 mg/l). Resistance to nalidixic acid coupled with a decreased susceptibility to ciprofloxacin (MIC 0.25-1.0 mg/l) was more common, with 14% of isolates showing these properties. Resistance to third-generation cephalosporins was rare with only 0.6% of isolates resistant to cefotaxime. In all countries multiple resistance was most common in Salmonella enterica serotype Typhimurium, with 51% of isolates multiresistant in total. In England and Wales multiple resistance was also prevalent in S. Virchow and S. Hadar, whereas in other countries multiple resistance was common in serotypes such as S. Blockley.

Resuscitation of Salmonella enterica serovar typhimurium and enterohemorrhagic Escherichia coli from the viable but nonculturable state by heat-stable enterobacterial autoinducer.

Salmonella enterica serovar Typhimurium and enterohemorrhagic Escherichia coli were stressed by prolonged incubation in water microcosms until it was no longer possible to observe colony formation when samples were plated on nonselective medium. Overnight incubation of samples in nutrient-rich broth medium supplemented with growth factors, however, allowed resuscitation of stressed and viable but nonculturable cells so that subsequent plating yielded observable colonies for significantly extended periods of time. The growth factors were (i) the trihydroxamate siderophore ferrioxamine E (for Salmonella only), (ii) the commercially available antioxidant Oxyrase, and (iii) the heat-stable autoinducer of growth secreted by enterobacterial species in response to norepinephrine. Analysis of water microcosms with the Bioscreen C apparatus confirmed that these supplements enhanced recovery of cells in stressed populations; enterobacterial autoinducer was the most effective, promoting resuscitation in populations that were so heavily stressed that ferrioxamine E or Oxyrase had no effect. Similar results were observed in Bioscreen analysis of bacterial populations stressed by heating. Patterns of resuscitation of S. enterica serovar Typhimurium rpoS mutants from water microcosms and heat stress were qualitatively similar, suggesting that the general stress response controlled by the sigma(s) subunit of RNA polymerase plays no role in autoinducer-dependent resuscitation. Enterobacterial autoinducer also resuscitated stressed populations of Citrobacter freundii and Enterobacter agglomerans.

Phage typing and PFGE pattern analysis as tools for epidemiological surveillance of Salmonella enterica serovar Bovismorbificans infections.

Some years ago, an increase in the number of sporadic cases and outbreaks of salmonellosis due to S. enterica serovar Bovismorbificans was observed in several European countries including Finland, Sweden, England/Wales, Austria, and Germany. In order to understand the recent spread of this serovar and to trace the route of infection back to its source, it was considered necessary to subtype S. Bovismorbificans isolates. Using phage typing (newly described here) and molecular fingerprinting (PFGE-pattern, plasmid profiles and ribotype) the isolates of European origin could be subtyped and compared to S. Bovismorbificans isolates that originated in overseas countries such as Australia, Thailand, India, etc. where this serovar was isolated more frequently. Significant clonal diversity was identified but some of the clonal types of S. Bovismorbificans dominated the epidemics and single cases in Europe as well as in overseas countries. The clonal identity among these isolates indicates an international distribution, new sources of infection, and highlights the urgent requirement for standardized laboratory based surveillance networks (e.g. Enter-Net). Moreover, it is suggested that strains of S. Bovismorbificans will continue to be of concern in public health and that phage typing together with PFGE typing can be applied as reliable and rapid tools for their future monitoring.

Occurrence of Salmonella enterica serovar Dublin in Austria.

In Austria, Salmonella enterica subsp. enterica serovar Dublin, a bovine-adapted serovar, rarely causes infections in humans. In 2000, Austria was within the European mean with an incidence of 0.1 per million inhabitants. Our data show that the vast majority of all serovar Dublin infections (human and non-human) can be traced epidemiologically to two districts in the Tyrol. This concentration of cases can be explained by a particularly traditional aspect of cattle farming in this area, the alpine pasture. There is an increased risk of cross infection due to the communal keeping of animals from various farms. Infected cattle are a source of infection for humans, and transmission usually occurs from eating beef and drinking cows milk. Using pulsed field gel electrophoresis and automated ribotyping, three out of five isolates from human infections could be traced to characteristic Tyrolean Dublin clones. Bacteriological screening for faecal carriage before the transfer of cattle from risk-herds to the alpine pastures and before the return from risk-pastures to the farms would be a possible starting point to prevent cross-contamination of large mixed herds and contamination of pasture through latently infected cattle. Appropriate research is necessary.