Global Genomic Epidemiology of Salmonella enterica Serovar Typhimurium DT104.

It has been 30 years since the initial emergence and subsequent rapid global spread of multidrug-resistant Salmonella entericaserovar Typhimurium DT104 (MDR DT104). Nonetheless, its origin and transmission route have never been revealed. We used whole-genome sequencing (WGS) and temporally structured sequence analysis within a Bayesian framework to reconstruct temporal and spatial phylogenetic trees and estimate the rates of mutation and divergence times of 315S Typhimurium DT104 isolates sampled from 1969 to 2012 from 21 countries on six continents. DT104 was estimated to have emerged initially as antimicrobial susceptible in ∼1948 (95% credible interval [CI], 1934 to 1962) and later became MDR DT104 in ∼1972 (95% CI, 1972 to 1988) through horizontal transfer of the 13-kb Salmonella genomic island 1 (SGI1) MDR region into susceptible strains already containing SGI1. This was followed by multiple transmission events, initially from central Europe and later between several European countries. An independent transmission to the United States and another to Japan occurred, and from there MDR DT104 was probably transmitted to Taiwan and Canada. An independent acquisition of resistance genes took place in Thailand in ∼1975 (95% CI, 1975 to 1990). In Denmark, WGS analysis provided evidence for transmission of the organism between herds of animals. Interestingly, the demographic history of Danish MDR DT104 provided evidence for the success of the program to eradicate Salmonellafrom pig herds in Denmark from 1996 to 2000. The results from this study refute several hypotheses on the evolution of DT104 and suggest that WGS may be useful in monitoring emerging clones and devising strategies for prevention of Salmonella infections.

Characterization of the emerging Salmonella 4,[5],12:i:- in Danish animal production.

The monophasic Salmonella variant with the antigenic formula Salmonella 4,[5],12:i:- has emerged in the last decade as one of the main serotypes related to human salmonellosis. In the present study, a collection of 94 isolates of the S. 4,12:i:- and S. 4,5,12:i:- coming from Danish farm animals, swine (86), cattle (7), and poultry (1), with well-defined identification was further typed by polymerase chain reaction serotyping, phage typing, and molecular typing (polymerase chain reaction and multilocus variable-number tandem-repeat analysis [MLVA]). Moreover, the determination of antimicrobial resistance pattern of each isolate was tested. In 68 of the isolates the fljB gene was absent (i.e., they were true monophasic strains), whereas in 26 isolates, the gene was present despite the fact that the isolates did not express it. The results clustered the isolates in three main pulse-types. The predominant cluster was compatible with the previously described pattern STYMXB.0131. All the isolates included in this cluster lacked the fljB gene, and all the isolates except one belonged to phage type DT 193 with the AMP-STR-SMX-TET resistance pattern. MLVA analysis divided the clusters in several MLVA profiles previously reported by other studies. Finally, antimicrobial resistance and multiresistance was frequent, although no resistance was detected in critical compounds: fluoroquinolones and cephalosporins. The present study demonstrates the presence of monophasic Salmonella Typhimurium-like strains in Danish food animal production with well-characterized clones that are described by previous studies, demonstrating the emergence and spread of this serotype in Denmark.

Salmonella Typhimurium Level in Mealworms (Tenebrio molitor) After Exposure to Contaminated Substrate.

Findings of viable Salmonella spp., which are important foodborne pathogens, are seemingly not reported in mealworms (Tenebrio molitor) for feed and food. Still, the bacterial load of mealworms is naturally high and includes members of the Enterobacteriaceae family to which Salmonella belong. This indicates that Salmonella may be able to thrive in mealworms if introduced into the production. Therefore, this study aimed to assess the quantitative level of Salmonella enterica serovar Typhimurium (ST) in mealworms over a 14-day course after exposure to substrate contaminated with ST levels from 1.7 to 7.4 log CFU/g at start (i.e., day 0). The level of ST found in larvae was below the quantitative detection level (1 or 2 log CFU/g) on day 1 in larvae exposed to contamination levels of 1.7, 3.4, and 3.6 log CFU/g opposed to contamination levels of 5.4, 5.6, and 7.4 log CFU/g, respectively. The maximum level of ST detected in individual 1-g larvae samples was 5.8 log CFU/g, but the level varied among the triplicate samples from each sampling, and the highest average value was 5.3 ± 0.3. Beyond day 7, only larvae exposed to the contamination level of 7.4 log CFU/g were >1.0 log CFU/g in the triplicate samples. However, qualitative testing (10 g) showed the presence of ST in larvae until the end of the experiment on day 14 except for the lowest contamination level of 1.7 log CFU/g. Parallel testing of surface disinfected larvae indicated that some larvae may be ST-positive due to Salmonella residing on the surface only. Still, any detection of Salmonella is of concern from a food safety perspective. In substrate with contamination levels below 3.6 log CFU/g, the level of ST was below the quantitative detection limit within a few days. Still, ST was detected until the end of experiment on day 14 except for the lowest contamination level of 1.7 log CFU/g. This study indicates the importance of avoiding introduction of Salmonella into the production, e.g., via contaminated substrate in order to avoid Salmonella-positive larvae as they remained positive for at least 14 days (except at the lowest contamination level).

Ten years of antimicrobial susceptibility testing of Salmonella from Danish pig farms.

OBJECTIVES: This study analysed the trends in antimicrobial resistance in Salmonella serovars and phage types from pigs in Denmark from 1997 to 2006. METHODS: Salmonella isolates collected through the Salmonella surveillance programme in pigs were serotyped and phage-typed, and susceptibilities to the following antimicrobials were determined: ampicillin, chloramphenicol, gentamicin, nalidixic acid, colistin, streptomycin, sulphonamide, tetracycline and trimethoprim. RESULTS: No significant development of resistance occurred within the most important serovars, except Salmonella Typhimurium. A major decrease in Salmonella Typhimurium DT12 occurred from 46.5% in 1998 to 16.8% in 2006 while DT120, DT170 and DT104 increased. Throughout the study period, 80.9% of the DT12 isolates remained susceptible to the antimicrobials tested despite an increase in antimicrobial consumption in pigs during the period. In DT120, DT170 and DT104, only 20.1%, 33.1% and 23.0%, respectively, remained fully susceptible. CONCLUSIONS: The results support that the use of antimicrobial agents might select for multiple resistant clones and that this might be the driver of changes in antimicrobial resistance within a serovar, rather than an emergence of resistance within clones. The results of this study also support that susceptible serovars only slowly become resistant to the antimicrobials tested.

Characterization of Campylobacter phages including analysis of host range by selected Campylobacter Penner serotypes.

BACKGROUND: The predominant food borne pathogen in the western world today is Campylobacter. Campylobacter specific bacteriophages (phages) have been proposed as an alternative agent for reducing the burden of Campylobacter in broilers. One concern in relation to phage biocontrol is the narrow host range often displayed by phages. To identify the potential of phages as a Campylobacter reducing agent we needed to determine their infectivity on a panel of isolates representing the Campylobacter strains found in broilers as well as humans. RESULTS: In this study, Campylobacter phages were isolated from the intestines of broilers and ducks and from abattoir sewage. Twelve phages were investigated to determine their ability to infect the Campylobacter Penner serotypes commonly present in Danish poultry and patients with campylobacteriosis. A total of 89% of the Campylobacter jejuni strains and 14% of the Campylobacter coli strains could be infected by at least one of the bacteriophages. The majority of the phages infected the most common serotypes in Danish broilers (O:1,44; O:2; O:4-complex), but showed limited ability to infect 21 of the less frequent Campylobacter serotypes. Pulse field gel electrophoresis (PFGE) and restriction endonuclease analysis (REA) were used to characterize the phage genomes. Three categories of bacteriophages were observed. I: a genome size of approximately 194 kb and refractory to digestion with HhaI; II: a genome size of approximately 140 kb and digestible by HhaI; and III: a genome size undeterminable in PFGE. The categorization of the phages correlated with the host range patterns displayed by the phages. Six phages were subjected to transmission electron microscopy (TEM). They all belonged to the family of Myoviridae. CONCLUSION: We have characterized and identified the host range of 12 Danish Campylobacter phages. Due to their ability to infect the majority of the common serotypes in Denmark we suggest the phages can become an effective agent in the effort to reduce the incidence of campylobacteriosis in Denmark. This study provides the basis for future experiments in Campylobacter phages and knowledge for the selection of Campylobacter phages for biocontrol in broilers.

Campylobacter jejuni and Campylobacter coli in wild birds on Danish livestock farms.

BACKGROUND: Reducing the occurrence of campylobacteriosis is a food safety issue of high priority, as in recent years it has been the most commonly reported zoonosis in the EU. Livestock farms are of particular interest, since cattle, swine and poultry are common reservoirs of Campylobacter spp. The farm environment provides attractive foraging and breeding habitats for some bird species reported to carry thermophilic Campylobacter spp. We investigated the Campylobacter spp. carriage rates in 52 wild bird species present on 12 Danish farms, sampled during a winter and a summer season, in order to study the factors influencing the prevalence in wild birds according to their ecological guild. In total, 1607 individual wild bird cloacal swab samples and 386 livestock manure samples were cultured for Campylobacter spp. according to the Nordic Committee on Food Analysis method NMKL 119. RESULTS: The highest Campylobacter spp. prevalence was seen in 110 out of 178 thrushes (61.8 %), of which the majority were Common Blackbird (Turdus merula), and in 131 out of 616 sparrows (21.3 %), a guild made up of House Sparrow (Passer domesticus) and Eurasian Tree Sparrow (Passer montanus). In general, birds feeding on a diet of animal or mixed animal and vegetable origin, foraging on the ground and vegetation in close proximity to livestock stables were more likely to carry Campylobacter spp. in both summer (P < 0.001) and winter (P < 0.001) than birds foraging further away from the farm or in the air. Age, fat score, gender, and migration range were not found to be associated with Campylobacter spp. carriage. A correlation was found between the prevalence (%) of C. jejuni in wild birds and the proportions (%) of C. jejuni in both manure on cattle farms (R(2) = 0.92) and poultry farms (R(2) = 0.54), and between the prevalence (%) of C. coli in wild birds and the proportions (%) of C. coli in manure on pig farms (R(2) = 0.62). CONCLUSIONS: The ecological guild of wild birds influences the prevalence of Campylobacter spp. through the behavioural patterns of the birds. More specifically, wild birds eating food of animal or mixed animal and vegetable origin and foraging on the ground close to livestock were more likely to carry Campylobacter spp. than those foraging further away or hunting in the air. These findings suggest that wild birds may play a role in sustaining the epidemiology of Campylobacter spp. on farms.

Outbreak of Salmonella enterica serovar Typhimurium phage type DT41 in Danish poultry production.

Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) is one of the most prevalent serovars in Europe - where both poultry and poultry related products are common sources of human salmonellosis. Due to efficient control programs, the prevalence of S. Typhimurium in Danish poultry production is very low. Despite this, during the past decades there has been a reoccurring problem with infections with S. Typhimurium phage type DT41 in the Danish poultry production without identifying a clear source. In the end of 2013 and beginning of 2014 an increased isolation of S. Typhimurium DT41 was noted mainly in this production, but also in other samples. To investigate this is in more detail, 47 isolates from egg layers (n=5, 1 flock), broilers (n=33, 13 flocks), broiler breeding flocks and hatches (n=5; 2 flocks and 1 environmental hatchery sample), feed (n=1), poultry slaughter house (n=3, environmental sample and meat) were typed with multi locus variable number of tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) to investigate the epidemiology of the outbreak. Based on PFGE results isolates were divided into four groups (Simpson's index of diversity (DI)=0.24±0.15). Due to the low DI, PFGE was not sufficient to provide information to unravel the outbreak. Based on MLVA typing the DT41 (42/47 isolates) and the RDNC isolates (5/47) were split into nine groups (DI=0.65±0.14). When a maximum divergence at one locus was permitted these could be gathered into four groups. Using this criterion, combined with epidemiological information, a spread of one type from broiler breeders to broilers and further to the poultry slaughter house was plausible. In conclusion, although it could be concluded that a spread within the broiler production pyramid had taken place the source of the sudden increase of S. Typhimurium DT41 remains unclear. To investigate this in more detail, further studies using whole genome sequencing to obtain a higher discriminatory strength and including isolates from a longer period of time and from various sources are in progress.

Reappearance of Salmonella serovar Choleraesuis var. Kunzendorf in Danish pig herds.

Salmonella enterica serovar Choleraesuis is a porcine adapted serovar which may cause serious outbreaks in pigs. Here we describe outbreaks of salmonellosis due to S. Choleraesuis in four Danish pig farms in 2012-2013 by clinic, serology, and microbiology and compare the isolates to those of a previous outbreak in 1999-2000. The infection was in some herds associated with high mortality and a moderate to high sero-prevalence was found. In 2012-2013 the disease contributed to increased mortality but occurred concomitant with other disease problems in the herds, which likely delayed the diagnosis by up to several months. Nine isolates from the four farms in 2012-2013 and 14 isolates obtained from the outbreak in Denmark in 1999-2000 were subjected to typing using pulsed-field gel electrophoresis (PFGE). Seven isolates were selected for whole genome sequencing (WGS). The PFGE results of 23 isolates displayed five different profiles. The isolates from 2012 to 2013 revealed two distinct profiles, both different from the isolates recovered in 1999-2000. Two of the 2012-2013 farms shared PFGE profiles and had also transported pigs between them. The profile found in the two other 2012-2013 farms was indistinguishable but no epidemiological connection between these farms was found. Analysis of the number of single nucleotide polymorphisms (SNPs) from the WGS data indicated that the isolates from the farms in 2012-2013 were more closely related to each other than to isolates from the outbreak in 1999. It was therefore concluded that the infection was a new introduction and not a persistent infection since the outbreak in 1999. It may further be suggested that there were two or three independent rather than a single introduction. The re-introduction of S. Choleraesuis in Denmark emphasizes the importance of strict hygiene measures in the herds. Further investigations using WGS are now in progress on a larger collection of isolates to study clonality at European level and trace the origin of the infections.

Herd prevalence of Salmonella spp. in Danish pig herds after implementation of the Danish Salmonella Control Program with reference to a pre-implementation study.

The problems addressed are: (1) comparison of prevalences of Salmonella spp. in different herd types in the Danish pig population after implementation of the Danish Salmonella Control Program (DSCP), and (2) to make a reference to a study from 1993/1994 (pre-implementation) with a discussion of possible biases when diagnostic methods differ slightly. The objectives were to present the prevalences of Salmonella spp., Salmonella Typhimurium, and multiresistant S. Typhimurium DT104 in Danish pig herds in 1998. Further, to discuss how herd prevalences may be compared to a previous study.A bacteriological study in 1998 comprised: (a) a random sample of slaughter pig producing herds (N=1962); (b) a random sample of farrow-to-grower (sow) herds (N=305); and (c) all breeding and multiplying (genetic) herds (N=366). A previous bacteriological study on Salmonella presence in 1993/1994 served as a model for the present study. The results of the study were that multiresistant S. Typhimurium DT104 was detected in one herd producing slaughter pigs. The herd apparent prevalences (HAPs) of Salmonella spp. were 11.7, 16.7 and 11.4% in genetic, sow, and slaughter pig herds, respectively. The conclusion of the study was that prevalence of multiresistant S. Typhimurium DT104 was low in the examined slaughter pig herds. The herd true prevalence (HTP) of Salmonella spp. in pigs had declined from before the start of the DSCP in 1993/1994 to 4 years later (1998).

Prevalence, serotypes and resistance patterns of Salmonella in Danish pig production.

The objective of this paper is to analyse in further detail the Danish results of the EFSA baseline studies in slaughter pigs and breeding herds, and compare them with the results obtained in (1) the pre-implementation study that was carried out to establish the initial prevalence values in fattening herds as part of the Danish Salmonella control programme, and (2) the study performed four years later in breeding and finishing herds to obtain information about the prevalence in breeding farms and the status of the finishers after the first years of the National Salmonella Control Programme. In the slaughter pigs Salmonella was detected in a 7.4% of 1218 ileocaecal lymph nodes and on 3.2% of 438 carcasses examined. Among the breeding herds examined by floor faecal or swab samples 122 of 298 (40.9%) were positive in at least one of the ten samples collected. The most prevalent serotypes were Salmonella Typhimurium in finishers and Salmonella Derby in breeding herds while the most prevalent phage types of the S. Typhimurium isolates were DT 12 and DT 120. The antimicrobial resistance analysis yielded a 35.2% of the isolates from the slaughter pigs resistant to one or more antimicrobials while 19.3% were resistant to four or more antimicrobials. A significantly higher percentage of resistance to antimicrobials was found in the S. Typhimurium isolates (χ(2)=4.72, p=0.029), where 42.9% presented resistance to one or more compounds. In breeding herds, just S. Typhimurium and S. 4,5],12:i: - isolates were tested. As many as 56.8% of the S. Typhimurium-like strains positive breeding farms had resistant strains, while 27% had multidrug resistant strains. The distribution of the isolates in regions showed that S. Derby is at present the predominant serotype in breeding farms from most of the regions of the country.

Survival and transmission of Salmonella enterica serovar typhimurium in an outdoor organic pig farming environment.

It was investigated how organic rearing conditions influence the Salmonella enterica infection dynamics in pigs and whether Salmonella persists in the paddock environment. Pigs inoculated with S. enterica serovar Typhimurium were grouped with Salmonella-negative tracer pigs. Bacteriological and serological testing indicated that organic pigs were susceptible to Salmonella infections, as 26 of 46 (56%) tracer pigs turned culture positive. An intermittent and mainly low-level excretion of Salmonella (<100 cells g-1) partly explains why the bacteriological prevalence appeared lower than the seroprevalence. Salmonella persisted in the paddock environment, as Salmonella was isolated from 46% of soil and water samples (n=294). After removal of pigs, Salmonella was found in soil samples for up to 5 weeks and in shelter huts during the entire test period (7 weeks). Subsequent introduction of Salmonella-negative pigs into four naturally Salmonella-contaminated paddocks caused Salmonella infections of pigs in two paddocks. In one of these paddocks, all tracer pigs (n=10) became infected, coinciding with a previous high Salmonella infection rate and high Salmonella excretion level. Our results showed that pigs reared under organic conditions were susceptible to Salmonella infections (just like conventional pigs) and that Salmonella persisting in the paddock environment could pose an infection risk. A driving force for these infections seemed to be pigs with a high Salmonella excretion level, which caused substantial contamination of the environment. This suggests that isolation of animals as soon as a Salmonella infection is indicated by clinical symptoms of diarrhea could be a means of reducing and controlling the spread and persistence of Salmonella in outdoor organic pig production environments.

Salmonellae in avian wildlife in Norway from 1969 to 2000.

Postmortem records of wild-living birds in Norway with laboratory-confirmed findings of salmonella infection were summarized for the period from 1969 to 2000. Salmonella spp. were isolated from 470 birds belonging to 26 species. The salmonella-positive birds included 441 small passerines, 15 gulls, 5 waterfowl, 4 birds of prey, 3 doves, and 2 crows. The bullfinch (Pyrrhula pyrrhula) was by far the most frequently recorded species (54% of the cases). Salmonella enterica serover Typhimurium was recovered from all cases except from one hooded crow (Corvus corone), which yielded serovar Paratyphi-B var. Java. Variant O:4,12 comprised 96% (451 cases) of all serovar Typhimurium isolates, including all the passerines, while variant O:4,5,12 accounted for the remaining 4% (18 cases). The occurrence of salmonellae in small passerines showed a distinct seasonality, with a peak in February and March. Plasmid profile analysis of 346 isolates of serovar Typhimurium O:4,12 detected six profiles, of which two comprised 66 and 28% of the isolates, respectively. Phage typing of 52 randomly selected isolates of serovar Typhimurium O:4,12 from passerines detected four types: DT 40 (54%), U277 (35%), DT 99 (6%), and DT 110 (4%).