Distribution and function of plasmids in Salmonella enterica

Plasmids of Salmonella enterica vary in size from 2 to more than 200 kb. The best described group of plasmids are the virulence plasmids (50-100 kb in size) present in serovars Enteritidis, Typhimurium, Dublin, Cholerae-suis, Gallinarum, Pullorum and Abortus-ovis. They all encode spvRABCD genes involved in intra-macrophage survival of Salmonella. Another group of high molecular weight plasmids are plasmids responsible for antibiotic resistance. Since most of these plasmids are conjugative, besides storage of genetic information, they contribute to the spread of genes in bacterial populations. The low molecular weight plasmids are the last group of plasmids found in S. enterica. Some of them have been shown to increase resistance to phage infection due to the presence of restriction modification systems. Despite limited knowledge on their function, their presence or absence is frequently used for strain differentiation in epidemiological studies.     

Prevalence of the Salmonella plasmid virulence gene "spvD" in Salmonella strains from animals]

Strains of Salmonella isolated from animals in Germany (n = 878) were analysed for the presence of the spvD gene ("Salmonella plasmid virulence gene D") by DNA-DNA hybridization. The spvD gene was only detected in strains of serovars Typhimurium (93.3%), Enteritidis (97.1%), and Dublin (100%) as well as in two rough strains of Salmonella enterica. Salmonella isolates from mammals carried the gene more frequently (cattle 94.0%, horses 92.6%, pigs 73.7%) than those from birds (33.3%) or reptiles (4.5%). Due to its high prevalence in epidemiologically relevant salmonellae, the virulence factor spvD may represent a sensitive and specific target in various serovars for diagnostic or immunization strategies.     

Salmonella pathogenicity islands in host specificity, host pathogen-interactions and antibiotics resistance of Salmonella enterica

Salmonella enterica is a pathogen highly successful in causing a variety of gastrointestinal and systemic diseases in animals and humans. While some serovars of S. enterica are able to infect a broad range of host organisms, other serovars are highly restricted to specific host species. The colonization of hosts by S. enterica depends on the function of a large number of virulence determinants. The molecular analyses of virulence genes demonstrated that most of these loci are clustered within Salmonella Pathogenicity Islands (SPI). SPI1 and SPI2 each encode type III secretion systems (T355) that confer main virulence traits of S. enterica, i.e. invasion, enteropathogenesis and intracellular survival and proliferation. Further SPI encode factors that contribute to intracellular survival, different types of adhesins, or effector proteins of the SPI1-T3SS or SPI2-T3SS. The availability of genome sequences of several serovars of S. enterica also revealed serovar-specific SPI. In this review, the main characteristics of the currently known SPI are summarized with focus on their roles in various animal hosts and putative functions in human infections.     

Report on Salmonella isolates submitted to the German National Reference Laboratory for running analyses and tests on zoonoses (Salmonella) in the year 2009

The present report deals with Salmonella strains received at the German National Reference Laboratory for Salmonella (NRL-Salm) for routine diagnostic in the year 2009. Hence, the working group continues the previous report from Friedrich et al. (2010) about the documentation on the serovar distribution of Salmonella received at the NRL-Salm in the years 2004-2008. As in the recent years, most of the Salmonella strains originated from livestock and food. In the year 2009 the NRL-Salm received 4765 isolates, most of them (85,1 %) were routine diagnostic. Salmonella ser. Typhimurium, its monophasic variant S. enterica subspecies enterica serovar 1,4,[5],12:i:- and Salmonella ser. Enteritidis were the most prevalent serovars. The number of S. enterica subsp. enterica serovar 1,4,[5],12:i:- isolates increased in 2009, in comparison to the years 2004-2008, and became the second most prevalent serovar serotyped at the NRL-Salm.     

Epidemiology of Salmonella enterica serovars enteritidis and Typhimurium in animals and people in Scotland between 1990 and 2001

Two serovars of salmonella which are currently of particular importance in both human and animal infections are Salmonella enterica serovars Enteritidis phage type 4 (PT4) and Typhimurium definitive type 104 (DT104). This paper describes the trends in the relationships between the levels of infection of people and a range of farm animal species with these two serovars and explores some of the reasons behind them. In 1996, there was a peak of 520 reports of S Typhimurium DT104 infection in people in Scotland, but the number has decreased every year since, to 96 in 2001. In cattle the incidence of S Typhimurium DT104 also peaked in 1996, with 138 incidents, and it has similarly decreased every year to 2001 when there were 10 reported incidents. Similar declines have been observed in its incidence in sheep and pigs. In people the number of reports of S Enteritidis PT4 peaked in 1997 at 1684 and then declined to 457 in 2001. In chickens, the number of reports of S Enteritidis PT4 peaked in 1998 at 34 incidents, but no incidents were reported in the following three years.     

Deletion of sodCI and spvBC in Salmonella enterica serovar Enteritidis reduced its virulence to the natural virulence of serovars Agona, Hadar and Infantis for mice but not for chickens early after infection

Salmonella enterica serovars Typhimurium, Enteritidis, Dublin, Choleraesuis or Gallinarum can colonise liver and spleen in particular hosts while infections with serovars Infantis, Agona, Hadar, etc. are usually limited to gastrointestinal tract. Reasons for this behavior are unknown, although it has been shown that sodCI and spv genes exhibit a strict distribution between more and less virulent serovars and they influence Salmonella virulence. However to what extent the presence or absence of these genes is associated with the increased virulence of serovars which possess them has never been addressed experimentally. In this study we therefore first confirmed the exclusive association of spvB and sodCI genes with the former group of serovars. In the next step we removed these two genes from S. Enteritidis genome and compared the virulence of such a mutant with the virulence of S. Infantis, S. Agona and S. Hadar for chickens and highly sensitive Balb/C mice. Single strain infection showed that the deletion of these two genes from S. Enteritidis resulted in the reduction of its virulence for mice but not for chickens. Mixed infection further confirmed these observations and indicated that in mice but not in chickens the virulence of sodCI and spv mutant was reduced to the natural virulence of serovars Infantis, Agona and Hadar. Although sodCI and spv genes do not influence S. Enteritidis virulence for chickens directly, they may be of an indirect effect through the increased persistence of S. Enteritidis in mice and increased probability of the reintroduction of S. Enteritidis into poultry flocks.     

Salmonella enterica serovar Typhimurium and Enteritidis infection of pigs and cytokine signalling in palatine tonsils

Pigs are considered as one of the major sources of zoonotic strains of Salmonella enterica for humans. Out of many S. enterica serovars, S. Typhimurium dominates in pigs, however, in several countries in Central Europe, S. Enteritidis is also quite frequent in pig herds. In this study we therefore compared the colonisation of pigs with S. Typhimurium and S. Enteritidis. We found that 3 weeks after infection S. Enteritidis 147 colonised the intestinal tract in higher quantities but was shed in faeces in lower quantities than S. Typhimurium 17C10. In a second experiment we found out that S. Enteritidis 147 and its SPI-1 and SPI-4 mutants increased proinflammatory cytokine (IL-1β and IL-8) signalling in the ileum 5 days post infection. On the other hand, independent of SPI-1 or SPI-4, S. Enteritidis 147 suppressed expression of IL-18, MCP1, TLR2, CD86, IL-7, IL-10 and IL-15 in the palatine tonsils. The suppression of cytokine signalling may facilitate the initial colonisation of the palatine tonsils by Salmonella. Moreover, immune suppression may also influence pig resistance to opportunistic pathogens and Salmonella infection in pigs thus may become an issue not only in terms of pork contamination but also in terms of affecting the immunological status of pig herds.     

Identification of Salmonella spp. isolates from chicken abattoirs by multiplex-PCR

The present study was carried out to report the occurrence Salmonella spp., Salmonella Enteritidis, and Salmonella Typhimurium in chicken abattoirs. Samples of feces; feathers; scald, evisceration, and chiller water; and rinse water of non-eviscerated, eviscerated, and chilled carcass were collected from six chicken abattoirs. Salmonella isolates were identified by a multiplex-PCR using three sets of primers targeting the invA, pefA, and sefA gene sequences from Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. Salmonella spp. was detected in 10% (29/288) of the samples, whereas serovars Enteritidis and Typhimurium were identified in 62% (7/288), respectively. The results indicate the need to improve hygiene and sanitary standards in poultry slaughter lines, besides the education of food handlers and information to consumers.     

Molecular characterization, spread and evolution of multidrug resistance in Salmonella enterica typhimurium DT104.

Multidrug-resistant Salmonella enterica serovar Typhimurium phage type DT104 has emerged during the last decade as a global health problem because of its involvement in diseases in animals and humans. Multidrug-resistant DT104 strains are mostly resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracyclines (ACSSuT resistance type). The genes coding for such resistances are clustered on the chromosome. This paper reviews new developments in the characterization of S. enterica Typhimurium DT104, its chromosomal antibiotic resistance genes and their spread among other S. enterica Typhimurium phage types and other S. enterica serovars, the development of specific detection methods, virulence characteristics, and the evolution of multidrug-resistance with regard to the emergence of quinolone resistance.     

Distribution of integrons and SGI1 among antibiotic-resistant Salmonella enterica isolates of animal origin

In Salmonella enterica, resistance to antibiotics can be caused by the presence of SGI1, transposons or conjugative plasmids. In this study we were interested in the relative contribution of these genetic elements to the antibiotic resistance of S. enterica isolates collected within a single year in the Czech Republic from animal sources. Altogether 123 antibiotic-resistant isolates belonging to 16 different S. enterica serovars were classified into 3 groups according to the presence of SGI1 and the presence of integrons. The first group consisted of 62 strains in which neither SGI1 nor class 1 integron was detected. A high diversity among serovars and resistance phenotypes was found in this group. The second group consisted of 56 strains positive for both the SGI1 and class 1 integron, out of which 55 belonged to serovar Typhimurium and one to a nonmotile serovar [4,12] which harboured the SGI1-B variant. The third group comprised five strains which were positive for class 1 integron but negative for the SGI1. Sequencing of the integrons in these isolates identified integron with sat1 and aadA1 gene cassettes in S. Sandiego and S. Pullorum, dfrA1 and aadA1 gene cassettes in S. Typhimurium integron, and aadA21 gene cassette in S. Braenderub and S. Zanzibar.     

The distribution of Salmonella serovars in chicken and humans in Lithuania

We studied serovars of Salmonella strains isolated from chicken and humans in Lithuania over the period from 2000 to 2004. Salmonella strains were isolated and identified according to the techniques recommended by International Organisation for Standardization (Microbiology of Food and Animal Feeding Stuff--Horizontal Method for the Detection of Salmonella, 1998, ISO, Geneva). The per cent of infected flocks with Salmonella in separate years was between 1.01% and 3.2% during the period of investigation. The contamination rate of broiler legs and breasts was higher (2.36% and 4.25%) than that of wings (0.82%). Eighteen serovars of Salmonella were identified from the total 300 isolated samples. The most prevalent serovars in chicken were Salmonella Enteritidis, Salmonella Infantis and Salmonella Typhimurium. Other serovars such as Salmonella Montevideo, Salmonella Djugu, Salmonella Isangi, Salmonella Bovismorbificans, Salmonella Mbankada, Salmonella Hadar were detected only in one to two samples. In general, similar serovars of Salmonella were found in humans and chicken (S. Enteritidis and S. Typhimurium), although distinct serovars were found only in humans or only in chicken. Analysis of the distribution of Salmonella serovars in humans during the seasons of the year indicated that the highest incidence of Salmonella was in Summer and in the beginning of Autumn. Analysis of the distribution of serovars during the study period indicated that there is a shift over time in both humans and chicken.     

Identification of an aadA2 gene cassette from Salmonella enterica subsp. enterica Serovar derby

During a study on Salmonella enterica subsp. enterica serovar Derby from slaughter-age pigs in Brazil, two epidemiologically unrelated multi-resistant S. Derby isolates were found to carry a class 1 integron with a single gene cassette. Sequence analysis confirmed that this gene cassette harboured an aadA2 gene. The aadA2 gene codes for an aminoglycoside adenyltransferase, which mediates resistance to the aminoglycoside streptomycin and the aminocyclitol spectinomycin. Although aadA2 gene cassettes are widely distributed among Salmonella, database searches identified an AadA2 protein indistinguishable from that of S. Derby only in single isolates of S. enterica subsp. enterica Enteritidis from France and S. enterica subsp. enterica Typhimurium from Japan. Structural analysis of the 59-base element revealed at least one base pair difference between the 59-base element of the aadA2 cassette from S. Derby and any of the 59-base elements deposited in the databases.     

Sero types, phage types and antibiotic susceptibilities of Salmonella strains isolated from horses in The Netherlands from 1993 to 2000

We studied 232 Salmonella strains from horses with salmonellosis in The Netherlands, isolated in the period from 1993 to 2000 in order to provide insight in the dynamics of sero-, phage types (pt) and antibiotic susceptibilities over time. The strains were tested for susceptibility to seven antimicrobial agents using the agar diffusion method. In addition, the isolates were sero typed and Salmonella enterica subspecies enterica Typhimurium and Enteritidis strains were further phage typed. S. Typhimurium strains of phage type 506 and 401 (both classified as DT 104 in the English phage typing system) were additionally tested for their susceptibility to chloramphenicol (C), streptomycin (S) and sulfonamides (Su). Resistance was common against tetracycline and ampicillin. Most strains were susceptible to enrofloxacin (Enr) and ceftiofur (Cef). Resistance to tetracycline (T), kanamycin (K), ampicillin (A) and trimethoprim/sulfonamide (Sxt) combinations decreased from 1993 to 2000, whereas the resistance to gentamicin (G), ceftiofur and enrofloxacin was stable over time. S. Typhimurium was the predominant serovar and showed more (multiple) resistance compared to other Salmonella serovars. Sixteen different resistance patterns were found, with resistance to T alone and the combination of ACSSuT and AKSxtT being the most common. The multiresistant S. typhimurium phage type 506 (DT 104) was the most common phage type isolated from horses and most of these strains showed the pentadrug resistance pattern ACSSuT. The S. Typhimurium phage type 401 (DT 104) was also found frequently with an ASSuT resistance pattern. The most common S. Typhimurium phage types in horses corresponded with those found in humans, pigs and cattle in the same period in The Netherlands.     

Experimental reproduction of bovine Salmonella encephalopathy using a norepinephrine-based stress model

Neurological disease represents a sporadic but serious manifestation of bovine salmonellosis that is thought to be related to systemic infection. Salmonella enterica serovar Dublin (S. Dublin) is the serovar most associated with systemic infection in cattle, although reports of neurological disease associated with S. Dublin or any other serovar are rare and usually anecdotal. This study reports the involvement of three strains of S. enterica, serovars Saintpaul, Montevideo, and Enteritidis, in Salmonella encephalopathies. Encephalopathies were reproduced in calves using a norepinephrine-based stress model. Neurological signs were not observed in calves infected with control strains of S. enterica, including S. Dublin, or in calves infected with clinical strains in the absence of norepinephrine. Therefore, norepinephrine may play a role in Salmonella encephalopathies.     

Specific adhesion and invasion of Salmonella Enteritidis in the vagina of laying hens

Salmonella Enteritidis is the predominant serovar associated with egg-borne salmonellosis in humans. The colonization of S. Enteritidis in the vagina may play a role in the production of S. Enteritidis-contaminated eggs. In the first experiment, the in vitro adhesion of S. Enteritidis in vaginal and follicular explants was compared with that of S. Typhimurium by bacteriological isolation methods. The mean number of S. Enteritidis associated with vaginal explants was significantly (P < 0.05) higher than S. Typhimurium associated with vaginal explants and both serovars associated with follicular explants. In the second experiment, the in vitro adhesion and invasion of S. Enteritidis strains in the vaginal epithelium was compared with that of several strains of S. Agona, S. Infantis, S. Hadar, S. Heidelberg, S. Montevideo and S. Typhimurium, by immunohistochemical methods. The mean number of Salmonella in the vaginal epithelium depended on their lipopolysaccharide (LPS) type, with the rank order as follows: LPS type O9 (S. Enteritidis) > LPS type O4 (S. Agona, S. Typhimurium and S. Heidelberg) > LPS type O7 (S. Montevideo and S. Infantis) and LPS type O8 (S. Hadar). This rank order of Salmonella invasiveness is in accordance with the frequency of Salmonella outbreaks involving contaminated eggs. These findings suggest that S. Enteritidis has a higher ability to colonize the vaginal epithelium than other serovars, and the Salmonella LPS type may play an essential role in tropism of the reproductive tract.     

Comparative proteomic analysis of Salmonella enterica serovars Enteritidis, Typhimurium and Gallinarum

Salmonella enterica includes several related serovars which have different host ranges and cause diseases of different severities. However, their pathogenic potential is unknown, and it is not clear what mechanisms are activated or inhibited during adaptation to a specific host environment. Some proteins are involved in the mechanism of pathogenicity at a molecular level and provide the functional aspects that create the diverse phenotypes. To compare proteomic analyses of the total proteins of Salmonella Enteriditis (SE), Typhimurium (ST), and Gallinarum (SG), two-dimensional gel electrophoresis (2-DGE) was performed using a pH 4-10 immobilized pH gradient (IPG) strip, and some proteins were identified by mass spectrometry (MS). After staining the gels, the proteins that were expressed at 10-fold or higher levels compared to other spots on the gel were characterized. Some of the identified proteins were related to virulence, such as β-lactamase, RfbH protein, and shikimate kinase. Additionally, there was a high level of variation between serovars despite the similarities in the expression patterns. Furthermore, this study shows that 2-DGE combined with MS is a useful tool for identifying proteins differentially expressed between serovars with different host ranges and pathogenic potential.     

Detection and identification of Salmonella Typhimurium in bovine diarrhoeic fecal samples by immunomagnetic separation and multiplex PCR assay

The aim of this study was to use the immunomagnetic separation (IMS) test plus a multiplex polymerase chain reaction (m-PCR) assay to detect Salmonella at genus level and also for the identification of Salmonella enterica serovar Typhimurium in bovine diarrhoeic fecal samples. In all, 400 bovine diarrhoeic fecal specimens were examined by conventional bacterial culture, IMS, and m-PCR. For m-PCR assay, four set primers were selected: 139-141, specific for inv-A gene of Salmonella spp and the RfbJ, FliC and FljB, specific for the rfbJ, FliC and fljB genes of Salmonella Typhimurium or other Salmonella serovars with similar antigenic properties. Thirty-three (8.25%) out of the 400 fecal samples were culture positive for Salmonella serovars. Of these, 66.7% (22 of 33) were Salmonella enterica serovar Typhimurium, and 9.1% (three of 33) were serovar Dublin. In the IMS + m-PCR, four amplified product (663, 526, 284 and 183 bp) were found in all specimens that had serovar Typhimurium (4,5,12:i:1,2), they corresponded, respectively, to the rfbJ, fljB, inv-A and Flic genes of this serovar. In serovar Dublin (1,9,12:g,p:-), Georgia (6,7:b:e,n,z(15)) and, Enteritidis (1,9,12;g,m:-) only one PCR product (284 bp) was amplified from the inv-A gene. In serovars Augustenborg (6,7:i:1,2) and Lindenburg (6,8:i:1,2) three positive bands (526, 284 and 183 bp) were amplified corresponding to the fljB, inv-A and Flic genes, respectively. In serovar Virchow (6,7:r:1,2) two amplified products (284 and 526 bp) from the inv-A and FliC genes were observed. In serovar Gloucster (1,4,12(27):i:1,w) three fragments (183, 284 and 663) from the FliC, inv-A and, rfbJ genes respectively, were observed. In the positive control as expected, four PCR products were amplified corresponding to the FliC, inv-A, fljB and rfbJ genes, respectively. In conclusion, the results of this study showed that detection of Salmonella at genus level with universal ST139-141 primers and identification of Salmonella Typhimurium by using specific primers of O4, H(2):1, 2 and H(1) antigens can potentially permit to more readily evaluate fecal and other types of samples for the presence of these organisms. Compared to bacteriological culture the combination of IMS and m-PCR resulted a faster method for the detection and identification of Salmonella at genus and serovar level by using of universal and specific primers.     

Application of nucleotide sequence of RNA polymerase beta-subunit gene (rpoB) to molecular differentiation of serovars of Salmonella enterica subsp. enterica.

To establish a molecular differentiation method for Salmonella enterica subsp. enterica, a hyper-variable region of RNA polymerase beta-subunit (rpoB) of S. enterica subsp. enterica (I), serotype Typhimurium, and Escherichia coli were investigated through comparison of nucleotide sequence of the region. The hyper-variable region was identified at 612-937 of the gene. After PCR amplification of the region in the 17 serotypes and two biotypes of serotype Gallinarum of S. enterica subsp. enterica (I), the nucleotide sequences of the region were determined and compared. All serotypes were distantly related to E. coli with 82.8-84.7% identities in nucleotide sequence while showing 96.6-100% identities with each other. According to the phylogenetic analysis based on the sequenced region with the neighbor-joining method, relatedness of biotype Gallinarum to serotype Enteritidis and biotype Pullorum was determined. Biotype Gallinarum was more closely related to serotype Enteritidis than biotype Pullorum. These results suggested that the 612-937 variable region of rpoB might be useful for molecular evolutionary analysis of serotypes of S. enterica subsp. enterica (I).     

Impact of Salmonella Typhimurium DT104 virulence factors invC and sseD on the onset, clinical course, colonization patterns and immune response of porcine salmonellosis

The present study was conducted to study the impact of the virulence factors invC and sseD of the two type III secretion systems of Salmonella enterica serovar Typhimurium (S. Typhimurium) on the pathogenesis of the porcine S. Typhimurium DT104 infection. For this purpose, two S. Typhimurium mutant strains with a disrupted invC gene of the Salmonella pathogenicity island 1 or with a disrupted sseD gene of the Salmonella pathogenicity island 2 have been studied in experimental infection of pigs. Twenty-two 7-week-old male hybrid pigs were either infected with one of the mutants or the wild-type S. Typhimurium DT104 strain. Each group was examined for clinical signs, Salmonella shedding rate and the specific antibody response. Survival and replication were evaluated by qualitative and quantitative determination of the colonization rate. The humoral and cellular immune responses were examined using isotype-specific ELISA and quantitative real-time PCR of IL-2, IL-4, IL-10, IL-12 and IFN-gamma. The results proved that both mutants had a lower virulence (with marked differences between both mutants) than the wild-type and that both virulence factors have importance in porcine salmonellosis. Only pigs infected with the wild-type S. Typhimurium DT104 exhibited typical clinical symptoms of salmonellosis like anorexia, vomiting, disturbed demeanour, fever and diarrhoea. Deletion of the invC gene resulted in a significantly reduced colonization rate. Interestingly, the mRNA expression of both type-1 and type-2 cytokines were significantly decreased in pigs infected with either the invC-mutant and the sseD-mutant strain.     

Early immune dynamics following infection with Salmonella enterica serovars Enteritidis, Infantis, Pullorum and Gallinarum: cytokine and chemokine gene expression profile and cellular changes of chicken cecal tonsils

Salmonella enterica subspecies enterica infection remains a serious problem in a wide range of animals and in man. Poultry-derived food is the main source of human infection with the non-host-adapted serovars while fowl typhoid and pullorum disease are important diseases of poultry. We have assessed cecal colonization and immune responses of newly hatched and older chickens to Salmonella serotypes Enteritidis, Infantis, Gallinarum and Pullorum. S. Enteritidis and S. Infantis colonized the ceca more efficiently than S. Gallinarum and S. Pullorum. Salmonella infection was also associated with increased staining for B-lymphocytes and macrophages in the cecal tonsils of infected birds. S. Enteritidis infection in newly hatched birds stimulated the expression of CXCLi1 and CXCLi2 chemokines in the cecal tonsils, while S. Gallinarum up-regulated the expression of LITAF. In older chickens, S. Enteritidis infection resulted in a significantly higher expression of CXCLi2, iNOS, LITAF and IL-10 while S. Pullorum appeared to down-regulate CXCLi1 expression in the cecal tonsils. Data from spleens showed either no expression or down-regulation of the tested genes.