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.     

Comparison of growth phase on Salmonella enterica serovar Typhimurium invasion in an epithelial cell line (IPEC J2) and mucosal explants from porcine small intestine

Salmonella Typhimurium DT104 is a zoonotic enteropathogen of increasing concern for human health. In this study, the influence of growth phase on invasiveness of a S. Typhimurium DT104 field isolate and two reference strains (SL1344 and ATCC 14028) was compared in IPEC J2 cells and mucosal explants from porcine ileum. Internalized bacteria were quantified by a gentamicin resistance assay. After 90 min of exposure to the apical aspect of epithelial monolayers or luminal surface of explants, internalization of all S. Typhimurium strains in mid-logarithmic phase of bacterial growth was comparable. Internalization of stationary phase bacteria was reduced relative to log phase bacteria, with DT104 exhibiting the greatest decrease. Growth phase-related differences in S. Typhimurium invasion are similar in porcine intestinal epithelial cells and mucosal explants, but may be greater in multidrug-resistant strains.     

Epidemiology and characterization of animal Salmonella enterica subspecies Enterica serotype typhimurium DT104 in Hungary

Reports on the internationally emerging significance of multiresistant zoonotic Salmonella in animals and man prompted studies to estimate the significance of multiresistant Salmonella enterica subspecies enterica serotype Typhimurium (S. Typhimurium) phage type DT104 of animal origin in Hungary. A collection of 231 strains (primarily of goose, turkey, poultry and porcine origin from the years 1997-1998) was tested for resistance against 7 selected antibiotics (ampicillin, chloramphenicol, enrofloxacin, nalidixic acid, streptomycin, tetracycline and sulphamethoxazole). Strains with resistance against 3 or more were defined as multiresistant. All strains were phage typed using Felix-Callow's S. Typhimurium phage typing system, and 91 of them (suspect DT104) were also typed according to Anderson's definitive typing (DT) system. In this study, 14% of animal strains from 1997-1998 was classified as DT104, for which turkey, pig and duck seemed to be the main carriers, and the multiresistant non-DT104 strains represented a further 6% of this collection. The prevalence of DT104 was highest among strains of turkey origin (50%), followed by strains of pig (29%), chicken (25%), duck (19%), and goose (3%) origin. The other DT104 related phage types (DT12 and U302) were only detected in the case of 4 strains (2 of porcine, and one each of turkey and of goose origin). The DT104 corresponded to the Felix-Callow types 2/3 or 2c/3 in each case, except in the case of 3 turkey strains where they corresponded to type 35/3. Nalidixic acid resistance was detected in all multiresistant turkey strains and in some of other animal origin but none of these strains were resistant to enrofloxacin. A retrospective analysis (based on the above relationship) indicated that S. Typhimurium strains corresponding to DT104 could be present and increase in the Hungarian farm animal population from about 2% to 20% between 1985 and 1990, in a manner similar to the emergence of human DT104, as reported elsewhere (Pászti et al., 2000). The 91 suspect DT104 strains were also tested for plasmid profile and for spvC gene indicating the presence of the large serotype specific plasmid (Ssp). No characteristic plasmid profile could be attributed to S. Typhimurium DT104. The serovar-specific large plasmid was detected by PCR for spvC in 100% of DT104 strains and in 77% of the non-DT104 strains. The virulence of two DT104 strains was tested in orally infected day-old chicks and compared with virulence of 4 non-DT104 strains. Higher colonizing virulence of DT104 strains could be established as compared to the other strains.     

Changes of multi-drug resistance pattern in Salmonella enterica subspecies enterica serovar typhimurium isolates from food-producing animals in Japan

A total of 153 isolates of Salmonella Typhimurium derived from food-producing animals in Japan between 2002 and 2005 were investigated for antimicrobial resistance and phage types related to definitive phage type 104 (DT104). The predominant resistance type was resistance to ampicillin, dihydrostreptomycin, kanamycin, and oxytetracycline in bovine (45.2%, 48/104) and resistance to dihydrostreptomycin and oxytetracycline in porcine isolates (58.7%, 27/48). DT104-related phage type was found in 32 of 104 bovine isolates, two of 48 porcine isolates, and one of eight isolates from poultry, showing that the proportion of the phage type in S. Typhimurium isolates from cattle and pigs significantly (P<0.01) decreased from 71.9% and 31.4% in 1999-2001 to 30.8% and 4.1% in 2002-2005, respectively.     

Cytokine response of porcine cell lines to Salmonella enterica serovar typhimurium and its hilA and ssrA mutants

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular bacterium which can infect and colonize pigs. After contact with enterocytes and macrophages, S. Typhimurium induces production of cytokines thus triggering the innate immune response. In this study we evaluated the cytokine response of two porcine cell lines, IPI-2I and 3D4/31, of epithelial or macrophage origins, respectively, to the wild-type S. Typhimurium and its hilA and ssrA mutants. We observed that the 3D4/31 cell line essentially did not respond to S. Typhimurium infection when a medium with foetal calf serum was used. However when the 3D4 cell line was incubated overnight in the presence of porcine serum, it efficiently responded to the wild-type strain and the ssrA mutant but not to the noninvasive hilA mutant as measured by mRNA quantification of TNF-alpha, IL-8 and GM-CSF by the real-time RT-PCR. In IPI-2I, all the cytokines were also induced by the wild-type S. Typhimurium and the ssrA mutant although the induction of TNF-alpha was lower than that induced by the wild-type strain. The hilA mutant was unable to induce any of the cytokines tested. The ssrA mutant can therefore be considered as more suitable for further vaccine development as the stimulation of innate immune response is important for animal protection against a challenge with virulent strains.     

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.     

Differential regulation of porcine beta-defensins 1 and 2 upon Salmonella infection in the intestinal epithelial cell line IPI-2I

Intestinal epithelial cells represent the first line of defence against pathogenic bacteria in the lumen of the gut. Besides acting as a physical barrier, epithelial cells orchestrate the immune response through the production of several innate immune mediator molecules including beta-defensins. Here, we establish the porcine intestinal cell line IPI-2I as a new model system to test the regulation of porcine beta-defensins 1 and 2. Gene expression of both defensins was highly upregulated by foetal calf serum components in normal growth medium. In serum-free medium, baseline expression remained low, but pBD-2 gene expression was increased 10-fold upon infection with Salmonella Typhimurium. Arcobacter cryaerophilus and Salmonella Enteritidis, pathogenic bacteria with comparable adhesion and invasion characteristics, failed to increase pBD-2 mRNA levels. Heat killed or colistin-treated Salmonella Typhimurium had no effect, showing that the upregulation of pBD-2 was dependent on the viability of the Salmonella Typhimurium. Gene expression of pBD-1 was regulated differently since an increase in pBD-1 mRNA was observed by Salmonella Enteritidis infection. We conclude that the IPI-2I cells can serve as a new model to study porcine beta-defensin regulation and that pBD-1 and pBD-2 are differentially regulated in this cell line.     

Prevalence of Escherichia coli O157:H7 prophage-like sequences among German Salmonella enterica serotype Typhimurium phage types and their use in detection of phage type DT104 by the polymerase chain reaction

A 1.6kb DNA fragment identified by random amplifiable polymorphic DNA differentiation (RAPD) from a Salmonella enterica serotype Typhimurium phage type DT104 isolate was used to investigate the prevalence of the region in 160 DT104 isolates, 83 other epidemiological important S. Typhimurium phage types and 20 strains selected from 17 other Salmonella serotypes. PCR screening tests using two different primer-sets derived from the RAPD fragment's nucleotide sequence showed that 76% of the 160 DT104 isolates investigated, including subtypes DT104A, DT104B, DT104B low, DT104H and DT104L, reacted positively. High sensitivity was shown for DT104 strains expressing at least the penta-resistance pattern ACSSuT (97% of 104 strains tested). DT104 susceptible strains showed only a sensitivity of 35% (17 strains tested). In contrast, 83% of the 83 strains from the other S. Typhimurium phage types reacted negatively. Strains from five out of the 17 other serotypes showed a positive signal with one primer-set. The other primer-set exhibited only a positive reaction with one S. Dublin isolate. The analysis of a 2415bp extended sequence revealed homologies to genes encoded by Escherichia coli O157:H7 prophages, suggesting that the described region contains genes of a prophage specific for DT104 and related phage types.     

Salmonella spp. in Bavarian liquid pig manure: occurrence and relevance for the distribution of antibiotic resistance

In a representative study, 380 manure samples of pig farms distributed all over Bavaria (Germany) were screened for the presence of Salmonella spp. at the time of manure application to soil using methods adapted to DIN EN ISO 6579. The isolates were tested according to DIN 58940 for their susceptibility towards 26 antimicrobial substances - such substances partly administered in animals, but mainly used in human therapy. Six out of 380 manure samples and, in addition, the only separated liquid manure sample examined, contained Salmonella isolates. Serotypes represented S. Typhimurium (n = 5), S. Derby (n = 1) and S. Infantis/S. Serogroup C1-(6,7:r:-)-form (each n = 1). Within the serovar S. Typhimurium, the most common phage type was DT104 (n = 3). All Salmonella isolates originated from 'big' farms (>30/45 sows on combined/breeding farms or >220 fattening pigs). Four out of six manure samples contained resistant isolates; all resistant Salmonellae were multi-resistant. Two out of three DT104-isolates showed the typical penta-resistance pattern often found in S. Typhimurium DT104, which was, furthermore, supplemented by other resistances. One Escherichia coli isolated together with a phage type DT104 S. Typhimurium also expressed the same penta-resistance pattern.     

Salmonella Genomic Island 1 (SGI1) and genetic characteristics of animal and food isolates of Salmonella typhimurium DT104 in Hungary

To study the genetic characteristics of DT104 strains of Salmonella Typhimurium and the prevalence of Salmonella Genomic Island (SGI1) in Hungary, 140 recent Salmonella strains of food and animal origin were examined. For the first time in Hungary, the SGI1 was found in 17 out of 59 S. Typhimurium isolates (all proven to be DT104 phage type). These 17 strains were then subtyped by pulsed-field gel electrophoresis (PFGE) into 6 pulsotypes which were less correlated with the geographic origin than with the animal species of origin.     

Integron content of Salmonella enterica serotype Typhimurium strains isolated in Hungary in the years 1997-1999

The integron content of 52 DT104/U302 phage type strains and 53 non-DT104/U302 strains of Salmonella enterica serotype Typhimurium (S. Typhimurium) was studied in PCR experiments using a 5'-CS/3'-CS primer pair (Lévesque et al., 1995). Forty-three out of 44 streptomycin- and/or ampicillin-resistant DT104 and related phage type strains were found to carry a 1 kb and/or 1.2 kb long integron. The other resistance markers did not affect the number and size of integrons; no integron-free multidrug-resistant (MDR) DT104 strains were found. The two large groups of DT104 strains (Felix-Callow's phage types 2 and 2c) proved to be identical in respect of integron patterns (IPs), supporting the views of those authors who consider DT104 a single clone. Strains of human and animal origin did not differ from each other in their IPs. Within the non-DT104 phage types, ampicillin- and/or streptomycin-resistant, integron-free MDR strains were also found. Based on amplicons varying between 290 and 3500 bp an IP system was suggested. The commonest amplicon sizes in non-DT104 strains were 1450 and 2050 bp. The IPs of DT104 strains and of non-DT104 strains containing an integron of 1 and 1.2 kb size were stable. In contrast, the IPs of other non-DT104 strains showed a varying degree of instability. Integron loss was frequently associated with spontaneous plasmid elimination and changes of R-type among the descendants of a given strain.     

Antimicrobial resistance in Salmonella enterica serovars Enteritidis and Typhimurium isolated from animals in Korea: comparison of phenotypic and genotypic resistance characterization

Fourteen and 22 each of Salmonella Enteritidis and Salmonella Typhimurium (S. Typhimurium) were isolated from animals from 1983 to 1999 in Korea and tested for their antibiotic resistance patterns, phage types and resistance gene patterns. S. Typhimurium isolates were highly resistant to streptomycin, sulfisoxazole and tetracycline, 95, 95 and 86%, respectively. The incidence of multiple antibiotic resistance (resistant to more than two drugs tested) of S. Typhimurium isolates was extremely high (100%) comparing to S. Enteritidis isolates (21%). Two of the five ACSSuT (ampicillin, chloramphenicol, streptomycin, sulfisoxazole and tetracycline) resistant type S. Typhimurium isolates were phage type definitive type 104 (DT104).For the detection of resistance related genes in S. Enteritidis and S. Typhimurium isolates, particularly ACSSuT type S. Typhimurium, antibiotic resistance genes, cmlA/tetR, bla(PSE-1) and bla(TEM), and genus Salmonella specific gene, sipB/C, were amplified using four pairs of primers in a hot-start multiplex polymerase chain reaction (PCR). Two Korean isolates of S. Typhimurium DT104 showed bla(TEM) amplicons instead of bla(PSE-1) for the ampicillin resistance and they were susceptible to florfenicol. The multiplex PCR used in this study was useful in characterization of multiple drug resistant Salmonella isolates, especially ACSSuT type S. Typhimurium, and identification of beta-lactamase gene distribution among Salmonella isolates.     

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.     

The dual role of wild phages for horizontal gene transfer among Salmonella strains

Salmonella bacteriophages seem to mediate horizontal transfer of virulence functions among Salmonella strains in two different ways: by general transduction and also by lysogenic conversion. The majority of wild phages isolated from Salmonella strains belong to the P22 like phages and were able to transduce. Our data show that the lysogenic conversion is generally accompanied by changes in the susceptibility to the typing phages used for epidemiological purposes. Similar phage type conversions to S. Typhimurium DT104 could be detected upon lysogenization with two other S. Typhimurium strains. For some S. Typhimurium strains the typical phage pattern is actually associated with alterations of virulence characteristics. For example, all tested wild type isolates of phage types DT49 and DT204 were found to be SopE phi-lysogens. The Anderson typing phages interfere with the prophages and/or cryptic phages and so the complex genetic short-term evolution can be demonstrated in the lab. This is one reason for the successful application of phage typing in Salmonella epidemiology since the 50s.     

Salmonella typhimurium DT104 in farmed rabbits

A total of 1,000 rectal samples were collected from rabbits coming from 25 rabbit farms in southern Italy. All samples were processed for isolation of Salmonella spp. by standard culture method based on the ISO 6579:2002 method. Salmonella spp. was isolated from 1/25 rabbit farms analyzed. In particular, four out of 1,000 rectal swab samples, taken from young rabbits, were serotyped as S. Typhimurium and phage typed as S. Typhimurium DT104. All the isolates were resistant to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (ACSSuT pentaresistance type). The findings of the present study suggest the rabbit as potential carrier of S. Typhimurium DT104.     

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.     

Salmonella (sero)types and their resistance patterns in pig faecal and post-mortem samples.

The purpose of this survey was to take stock of porcine Salmonella isolates derived from faecal and post-mortem samples over a 4-year period. Salmonella was isolated by direct inoculation on BGA(NO)-plates (faeces, intestinal content) or sheep blood agar (organs). Antimicrobial susceptibility was tested by the agar diffusion method. Salmonella was isolated in 4.2% of all porcine submissions received at the Animal Health Service. A total of 1305 salmonellae were isolated from a total of 1279 submissions from 1008 different herds. Salmonella Typhimurium was the most frequently isolated serotype (88%), and Salmonella Typhimurium DT104 was the most frequently isolated phagetype (17.2% of Salmonella Typhimurium). Resistance to antimicrobials occurred in 47.3% of all isolates, mainly those of the multiresistant phagetype DT104. Other pathogens were isolated in more than 50% of the submissions. In cases of clinical diarrhoea, multiple pathogens may be involved and therapy and preventive measures should be adjusted accordingly.     

Risk factors for clinical Salmonella enterica subsp. enterica serovar Typhimurium infection on Dutch dairy farms

Risk factors for outbreaks in 1999 of clinical Salmonella enterica subsp. enterica serovar Typhimurium infection on dairy farms were studied in a matched case-control study with 47 case farms and 47 control farms. All 47 case farms experienced a clinical outbreak of salmonellosis which was confirmed with a positive bacteriologic culture for serovar Typhimurium in one or more samples. Serovar Typhimurium phage type 401 and 506 (definitive type 104, DT104) were the most frequently isolated phage types (13 isolates). On most farms (66%), clinical signs were seen only among adult cows. The most frequently reported clinical signs were diarrhoea (in 92% of the farms) and depression (in 79% of the farms).Control farms were matched on region and had no history of salmonellosis. A questionnaire was used to collect data on case and control farms. The relationship between serovar Typhimurium status of the farm and possible risk factors was tested using conditional logistic regression. Significant factors in the final model were presence of cats on the farm (OR=0.06), purchase of manure (OR=21.5), feeding colostrum only from own dam (OR=0.08), a non-seasonal calving pattern (OR=25), unrestricted grazing of lactating cows (OR=0.07), and a high mean mowing percentage of pasture (OR=1.02).     

Evaluation of molecular typing methods for Salmonella enterica serovar Typhimurium DT104 isolated in Germany from healthy pigs

The discriminatory power of four different DNA based typing methods was tested for the molecular subtyping of Salmonella Typhimurium phage type DT104 isolates. German DT104 strains (n = 133) originating from slaughter pigs were analysed by plasmid profiling, and 32 of them by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes XbaI, SpeI or BlnI, random amplification of polymorphic DNA (RAPD) using 13 different primers and IS200 typing. A resulting subtyping scheme was obtained which is based on the most discriminatory power of the individual methods i.e. plasmid profiling and PFGE with all three enzymes. The index of discrimination obtained by the subtyping scheme was 0.909 closely approaching the maximum value of one. Although minor differences occurred in the molecular DNA pattern of single DT104 strains, a dominating subtyping pattern was observed confirming other studies which showed, that S. Typhimurium DT104 isolates are highly clonal.     

Salmonella enterica infections in Spanish swine fattening units

The present study is the first conducted in Spain to estimate the bacteriological herd prevalence of Salmonella enterica in fattening units and to describe the Salmonella serovar diversity on these farms using a sample representative of the entire swine population. For this purpose, 10 faecal samples were collected from 10 different pens containing pigs close to market weight in a total of 232 fattening units. Total sample size was proportionally distributed according to the fattener census in each of the regions of the country and all the samples were examined by culture of 25 g of faecal material. One hundred (43.1%) farms had at least one Salmonella-positive sample (95% CI: 37-49.1%). Salmonella enterica was detected in 290 (12.5%) pooled faecal floor samples (95% CI: 11.2-13.8%). The apparent herd prevalence of salmonellosis was similar among multi-site, finishing and farrow to finish farms. Overall, 24 different serovars were identified, with S. Typhimurium, S. Rissen and S. Derby being the most common both at herd and sample level. Results of phage typing were available for the 91 isolates of S. Typhimurium. A total number of 10 different phage types were identified, with DT 193 being the most frequent. Phage types DT 104, DT 104b and DT U302, which have been associated with several multi-resistant patterns, accounted for 23% and 29% of the Typhimurium total isolates or Typhimurium infected farms respectively.