Phage conversion in Salmonella enterica serotype Enteritidis: implications for epidemiology.

A model system for the study of phage conversion of Salmonella enterica serotype Enteritidis is reported. Temperate phages 1,2,3 and 6 from the phage typing scheme were used to convert several individually recognized phage types into others. Phage type 4 was converted to PT8, PT6a to PT4, PT6a to PT7, PT13 to PT13a and PT15 to PT11; some new phage lysis patterns were also detected. This model was used to examine the relationships between phage types within a previously defined clonal lineage, SECLIII, to establish whether or not Enteritidis like Salmonella enterica serotype Typhi and Salmonella enterica serotype Paratyphi B possessed type determining phages. We were able to convert PT1 to PT20, and PT15 to PT11.     

Increasing quinolone resistance in Salmonella enterica serotype Enteritidis

Until recently, Salmonella enterica serotype Enteritidis has remained sensitive to most antibiotics. However, national surveillance data from Denmark show that quinolone resistance in S. Enteritidis has increased from 0.8% in 1995 to 8.5% in 2000. These data support concerns that the current use of quinolone in food animals leads to increasing resistance in S. Enteritidis and that action should be taken to limit such use.     

Occurrence of Salmonella enterica serotype Enteritidis phage types in the Slovak Republic

Phage typing of 741 isolates of Salmonella enterica serotype Enteritidis from the slovak Republic in 1995 has been carried out using the scheme of Ward and colleagues (1987). 202 strains (51 isolated from food) were from 9 outbreaks, 536 isolates were from sporadic cases and 3 isolates were from nosocomial infections of new-born babies. 704 isolates (95%) from all sources were typeable and belonged to 7 different phage types (PTs). PT8 was the phage type most frequently identified (72.6%). Other epidemiologically relevant phage types were PT2 (8.5%), 4 (7.9%) and 1 (4.2%). With an incidence of 1.3--0.1% isolates of PTs 21, 6, 9 were found. 26 (3.5%) isolates were designated RDNC and 11 (1.5%) were untypeable.     

Pheno-genotyping of Salmonella enterica serotype Enteritidis isolates identified in Sicily during a reemergence period

After an upward trend paralleling that occurring in most European countries, including Italy, since October 2002 Salmonella enterica serotype Enteritidis (S. Enteritidis) has again gained the first position among outbreak and sporadic human isolates of Salmonella in Sicily. Because phage typing of S. Enteritidis has many technical and epidemiological limitations and molecular methods have proved to be poorly discriminative for this organism, multiple typing, using phage typing together with pulsed field gel electrophoresis (PFGE) and plasmid profiling on a sample of fifty human and poultry isolates identified during the period October 2002 to May 2003 in Sicily, was chosen as the most valuable strategy to explore key features of this new epidemic wave. Although the limited number of strains imposes a cautious interpretation of the results, an apparently increasing phage type heterogeneity has emerged with rise in PT6 as the more striking event. While PFGE has confirmed the findings by other authors about the close genetic homogeneity between PT4 and PT6, plasmid profiling has provided discriminative patterns for PT6 strains. Combined phenotypic and genotypic profiles are necessary for epidemiological studies and public health investigations on S. enteritidis.     

Specific detection of Salmonella enterica serotype Enteritidis using the polymerase chain reaction.

An assay was developed for the specific detection of Salmonella enterica serotype Enteritidis, using a novel application of the polymerase chain reaction (PCR). This PCR assay is based on the mismatch amplification mutation assay, an allele-specific reaction, and can discriminate Enteritidis from all other salmonella. PCR primers were selected to amplify a 351-base pair (bp) DNA fragment from the salmonella plasmid virulence A (spv A) gene of Enteritidis. A single base difference at position 272 is present between the nucleotide sequence of the spvA gene of Enteritidis and other salmonellae. The downstream PCR primer, that encompasses position 272 of the Enteritidis spvA gene, was designed to contain a single base mismatch at the penultimate position, resulting in a 1-base mismatch with Enteritidis and a 2-base mismatch with other salmonellae that harbour the virulence plasmid. The upstream primer was completely homologous with the region immediately 5'' to the spvA gene. When these primers were used and the annealing and extension reactions were performed at the same temperature, the PCR assay was specific for Enteritidis; no PCR product was detected for 40 other serotypes and 28 different genera examined. In pure culture, 120 colony forming units (c.f.u.) could be detected; a PCR product was observed from template derived from a 5 h enrichment broth culture of chicken seeded with 1 c.f.u. per gram of Enteritidis. This PCR assay is specific, reproducible, and less time consuming than the standard bacteriological methods used to detect Enteritidis.     

Levels of virulence are not determined by genomic lineage of Salmonella enterica serotype Enteritidis strains

Mouse virulence and the ability to adhere to, and invade cultured MDCK cells were investigated in 38 phage type reference strains of Salmonella enterica serotype Enteritidis and correlated with genomic lineage. The genomic lineage of 11 of the strains was determined in the present study; one IS200 and one ribotype pattern that had not been reported previously were observed. Log c.f.u. in the spleen 10 days post intraperitoneal (i.p.) infection with 3x10(3) bacteria (logVC10) varied between 2.9 and 8.7. The reference strains of PT7 and PT23 were found to be semi-rough and were of low virulence. All other strains possessed smooth LPS. Within each of the two major clonal lines, as well as among phage types outside these, both highly virulent and moderate to low virulent strains were present. While all strains of PT1, PT2 and PT8 were highly virulent, low virulent strains were detected in PT4 and PT13. The ability to adhere to, and invade MDCK cells varied between phage types (adherence between 13 and 61% of the inocula and invasion between 4 and 151% of the adherent cells). The results of the cell culture experiments did not correlate with the results of mouse virulence tests. No correlation between clonal lineage and virulence was found within S. Enteritidis. It seems most likely that some strains have lost some of the essential factors enabling this serotype to cause successful systemic infection.     

Virulence of Salmonella enterica serotype Enteritidis aflagellate and afimbriate mutants in a day-old chick model.

Certain fimbriae and the flagellae of Salmonella enterica serovar Typhimurium have been shown to contribute to attachment and invasion of gut epithelium in the murine typhoid infection model and to contribute to pathogenesis in the chick. However, little is known of the role these organelles play in Enteritidis poultry infections and, to study this, day-old chicks were dosed orally in separate experiments with defined multiply afimbriate and/or aflagellate mutant strains of Enteritidis. The colonization and invasion characteristics of each mutant were compared with those of the isogenic wild type strain by the determination of the number of bacteria recovered from livers and spleens at known time points post infection. Compared with wild type Enteritidis, a mutant unable to express flagella but retaining the genetic potential to express fimbriae was recovered post mortem from livers and spleens in significantly reduced numbers compared to the isogenic wild-type at all time points post infection (P < 0.001). Conversely, a flagellate but multiply afimbriate mutant (defective for the elaboration of five different fimbrial types) and a flagellate but non-motile 'paralysed' mutant were recovered from livers and spleens in similar numbers to the wild-type. The data suggested that Enteritidis flagella, but not fimbriae, played an important role in pathogenesis in the chick model and that the flagellar apparatus itself and not motility per se contributed significantly to this role.     

Molecular characterization of Salmonella enterica serotype Enteritidis isolates from humans by antimicrobial resistance, virulence genes, and pulsed-field gel electrophoresis

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major serovar associated with human salmonellosis. A total of 425 clinical S. Enteritidis isolates of human origin were collected between June 2009 and September 2010 from North Carolina. The isolates were further characterized for antimicrobial susceptibility, antimicrobial resistance coding determinants, virulence genes, and fingerprint profiles to determine whether they were similar or different to the S. Enteritidis strain responsible for the human outbreak due to consumption of contaminated eggs. Ten different antimicrobial resistance phenotypes were observed with the highest frequency of resistance exhibited to ampicillin (n=10; 2.35%). The isolates were predominantly pansusceptible (n=409; 96.23%); however, seven isolates were multidrug resistant (MDR; i.e., resistant to three or more antimicrobials). Extended spectrum β-lactamase (ESBL) coding genes (bla(TEM) and bla(PSE)) were detected in the ampicillin-resistant isolates, whereas a single MDR isolate tested positive for class 1 integron (1 kb). The majority of the isolates (n=422; 99.3%) carried the invA, mgtC, stn, sopB, sopE1, and sefA virulence genes. However, 37 (8.7%) and 46 (10.82%) S. Enteritidis isolates tested negative for the plasmid encoded genes spvC and rck, respectively. Pulsed-field gel electrophoresis (PFGE) typing of 118 S. Enteritidis isolates by restriction enzymes XbaI and BlnI resulted in seven clusters, each with a discriminatory index (DI) of 0.715 and 0.785, respectively. The combination of XbaI-BlnI patterns generated a dendrogram with 14 clusters and a higher DI of 0.914. The PFGE profile of 80 isolates matched 100% with the S. Enteritidis strain that has been cited for the recent outbreak in the United States due to consumption of contaminated eggs. In conclusion, we identified a genotypic similar S. Enteritidis population in our study based on antimicrobial susceptibility, virulence gene, and PFGE fingerprint profiles.     

An outbreak of human salmonellosis caused by ampicillin-resistant Salmonella enterica serovar Enteritidis PT13 in the Czech Republic

In summer 2004, an outbreak caused by Salmonella enterica serovar Enteritidis phage type 13 (S. Enteritidis PT13) was recorded in the Czech Republic. As well being a relatively rare phage type the strain was also ampicillin resistant. Outbreak (n=39) and pre-outbreak isolates (n=13) were characterized by pulsed-field gel electrophoresis (PFGE), beta-lactamase gene polymerase chain reaction and plasmid profile. The majority of outbreak isolates (n=37) were identical in XbaI PFGE profile, and two other outbreak isolates each differed from this profile by one or two fragments respectively. The pre-outbreak isolates were uniform in PFGE profile but distinct from the outbreak strain. Ampicillin resistance was confirmed to be encoded by the blaTEM gene located on the TnA transposon. This gene was readily transferable to a S. Enteritidis recipient strain and was associated with the transfer of a 200-kb plasmid. Our results indicate that all S. Enteritidis PT13 tested from 2004 belonged to a single outbreak strain which prior to 2004 had not been recognized in the Czech Republic.     

World Health Organisation--supervised interlaboratory comparison of ELISAs for the serological detection of Salmonella enterica serotype Enteritidis in chickens.

A collaborative exercise, supervised by the World Health Organisation, was set up to compare ELISAs used for the serological detection of Salmonella enteritica serotype Enteritidis in chickens. The aim was to ascertain how far agreement could be reached on the interpretation of optical density readings for high titre, intermediate titre and low titre sera. Two sets of sera were sent to 14 participants. The first set compared high, medium and low titre sera raised in specified-pathogen-free and commercial broiler breeder chickens. The second set comprised 20 sera of different antibody titres raised in commercial birds reared under laboratory conditions and sent blind. Both indirect and double-antibody sandwich blocking ELISAs were used with a number of different detecting antigens. With a few exceptions good agreement was reached on the interpretation of results obtained from high and low titre sera from the optical density obtained with a single serum dilution. Differences were observed in the interpretation of medium titre sera. The results suggested that most ELISAs produce reasonably comparable results and that practical problems may arise from interpretation of the results mainly as a result of the choice of the criteria used for differentiating sera obtained from infected and uninfected chickens. These problems are discussed.     

Surveillance of antimicrobial resistance of Salmonella enterica serotype Typhi in seven Asian countries

Two hundred and four Salmonella enterica serotype Typhi (S. Typhi) isolates were collected from seven Asian countries during 2002-2004. Multidrug-resistant S. Typhi (resistant to > or = 3 antibiotics) was detected in 84 (41.2%) isolates and 142 (69.6%) showed reduced susceptibility to ciprofloxacin (minimum inhibitory concentration=0.125-1.0 mg/l). This study highlights the worsening situation of antimicrobial resistance of S. Typhi in Asia.     

Molecular epidemiological investigation of a diffuse outbreak caused by Salmonella enterica serotype Montevideo isolates in Osaka Prefecture, Japan

In Osaka Prefecture, Japan, three foodborne outbreaks were caused by Salmonella enterica serotype Montevideo in rapid succession between September 2007 and May 2008. Further, Salmonella Montevideo was also isolated from several sporadic diarrhea patients and asymptomatic carriers examined during approximately the identical period. To investigate the relatedness of the isolates, we performed antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) analysis, and multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) for 29 Salmonella Montevideo isolates obtained in this region between 1991 and 2008. Although antimicrobial susceptibility tests had low discriminatory power, PFGE patterns revealed 17 unique types with <90% similarity in combined analyses involving XbaI and BlnI. Moreover, we detected three VNTR loci that were useful to genotype Salmonella Montevideo isolates, with our method ultimately classifying the isolates into 11 MLVA types based on differences in repeat unit number in each examined locus. Six isolates obtained from patients of two separate foodborne disease outbreaks, one sporadic patient, and three different carriers between 2007 and 2008 had nearly identical PFGE patterns and were classified into the identical MLVA type; further, the isolates with this PFGE and MLVA pattern appeared only at that time between 1991 and 2008. These data strongly suggest that genetically identical Salmonella Montevideo strains may have caused the 2007 and 2008 outbreaks in Osaka Prefecture. Our results demonstrate that PFGE using XbaI and BlnI is useful for discriminating between Salmonella Montevideo isolates, even within a limited area, and reconfirm that continuous epidemiological surveillance for bacterial intestinal infections such as salmonellosis may be useful to not only monitor changes in the genetic diversity of isolates, but to also detect diffuse outbreaks.     

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.     

Characterization of Salmonella enterica serotype typhimurium in the Czech Republic: phage types, antimicrobial and plasmid profiles

In this study a collection of 547 S. Typhimurium strains isolated in the years 2000 and 2001 both of the human and non-human origin were analysed. 21 different phage types were detected, the most frequent one was DT104 (46%) followed by DT141 (28%) and DT68 (3%). Resistance to one or more antimicrobial agents was found mainly in DT104 (77.4%). S. Typhimurium isolates resistant to 5 and more antimicrobial agents were found in three phagetypes DT104 (57%), DT120 and DT155. Plasmid profiling of DT104 isolates showed 10 different profiles. Pattern A found in 30.5% of tested strains was predominant and carried serovar specific plasmid and one additional small plasmid of approx. 2.5 kb.     

Role of electronic data exchange in an international outbreak caused by Salmonella enterica serotype Typhimurium DT204b

From July through September 2000, patients in five European countries were infected with a multidrug-resistant strain of Salmonella Typhimurium DT204b. Epidemiologic investigations were facilitated by the transmission of electronic images (Tagged Image Files) of pulsed-field gel electrophoresis profiles. This investigation highlights the importance of standardized protocols for molecular typing in international outbreaks of foodborne disease.     

Novel surveillance of Salmonella enterica serotype Heidelberg epidemics in a closed community

Pathogen and disease surveillance and control represent important public health priorities in high-density and high-risk populations such as nursing homes, cruise ships, military bases, hospitals, and prisons. Reportable disease investigations, along with syndromic surveillance, have been used to identify and characterize outbreaks in their early stages. In this study, we provide evidence that ongoing wastewater monitoring could be used to supplement these traditional methods in at-risk closed communities. During 2003-2005, a systematic and regularly timed human and farm-animal wastewater sampling scheme existed in several geographically distinct locations of a multisite population in Texas. In early July 2003, an outbreak of gastroenteritis caused by Salmonella enterica serotype Heidelberg occurred in the human population at one site. Wastewater samples from the weeks before, during, and after the outbreak were tested for the pathogen. Selective culture, serogrouping, and serotyping techniques as well as real-time polymerase chain reaction and pulsed field gel electrophoresis were used to detect and characterize the Salmonella Heidelberg in each sample. The ability to detect the causative pathogen of an outbreak while it circulates in the host populations prior to and after an outbreak, as well as during the outbreak peak, suggests that wastewater could be used as a supplemental disease surveillance tool. To further explore this possibility, two subsequent outbreaks of uncharacterized gastroenteritis in additional locations were also investigated using wastewater samples.     

Analysis of the FoodNet case-control study of sporadic Salmonella serotype Enteritidis infections using persons infected with other Salmonella serotypes as the comparison group

Use of well persons as the comparison group for laboratory-confirmed cases of sporadic salmonellosis may introduce ascertainment bias into case-control studies. Data from the 1996-1997 FoodNet case-control study of laboratory-confirmed Salmonella serogroups B and D infection were used to estimate the effect of specific behaviours and foods on infection with Salmonella serotype Enteritidis (SE). Persons with laboratory-confirmed Salmonella of other serotypes acted as the comparison group. The analysis included 173 SE cases and 268 non-SE controls. SE was associated with international travel, consumption of chicken prepared outside the home, and consumption of undercooked eggs prepared outside the home in the 5 days prior to diarrhoea onset. SE phage type 4 was associated with international travel and consumption of undercooked eggs prepared outside the home. The use of ill controls can be a useful tool in identifying risk factors for sporadic cases of Salmonella.     

Vaccination of chickens with Salmonella Pathogenicity Island (SPI) 1 and SPI2 defective mutants of Salmonella enterica serovar Enteritidis

In this study we were interested in the vaccine potential of two attenuated mutants of Salmonella enterica serovar Enteritidis for poultry. The first mutant was attenuated by the removal of the whole Salmonella Pathogenicity Island 1 (SPI1) and the second mutant was devoid of the whole SPI2. These 2 mutants were used for oral vaccination of 2 chicken lines; Lohmann Brown and ISA Brown. Chickens were vaccinated orally on day 1 of life, revaccinated on day 21 and challenged on day 42. The challenge was performed either orally or intravenously. Despite a slightly different response between the two chicken lines, both the mutants gave protection to poultry against S. Enteritidis challenge as documented by findings such as the bacterial counts in tissues, spleen weight, antibody production and cytokine response (namely IL-17 and IL-22). When the 2 mutants were compared, vaccination with the SPI1 mutant proved to be more effective in the protection of poultry against S. Enteritidis challenge than the vaccination with the SPI2 mutant. On the other hand, vaccination with the SPI2 mutant stimulated a slightly higher antibody production and such a mutant might therefore be a better choice if Salmonella is used as a vector for the delivery of heterologous antigens with a desired stimulation of the humoral part of the immune system.     

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.