Gitte Sørensen

Tandem Repeat Analysis for Surveillance of Human Salmonella Typhimurium Infections

Multiple-locus variable-number tandem repeats analysis improves surveillance and outbreak investigations.

International Spread of Multidrug-resistant Salmonella Schwarzengrund in Food Products

This serovar was isolated from persons, food, and food animals in Thailand, Denmark, and the United States.

International Spread of blaCMY-2-Mediated Cephalosporin Resistance in a Multiresistant Salmonella enterica Serovar Heidelberg Isolate Stemming from the Importation of a Boar by Denmark from Canada

Salmonella isolates resistant to oxyiminocephalosporins due to the production of extended-spectrum β-lactamases (ESBLs) have emerged worldwide since 1992 (2). This has caused concern since cephalosporins are drugs of choice for the treatment of salmonellosis in children, to whom fluoroquinolones must not be administered. Different blaSHV, blaTEM, blaCTX, and blaCMY genes have encoded ESBL production in Salmonella (5, 8, 9). The same genes have been shown to encode resistance in different countries, which could indicate a global spread of these genes. In Denmark ESBL-producing isolates of Salmonella or other gram-negative bacteria have not previously been isolated from food-producing animals. However, in August 2003 the first ESBL-producing Salmonella isolate was isolated from the intestine of a boar imported from Canada. The boar was imported in May 2003 by a Danish breeding station and suddenly died on 4 August. The isolate was serotyped as Salmonella enterica serovar Heidelberg and was examined for antimicrobial susceptibility by MIC determinations (6). The β -lactam resistance was not transferable to E. coli recipients, whereas transfer was obtained in high frequencies to nalidixic acid-resistant S. enterica serovar Heidelberg, serovar Typhimurium, and serovar Dublin recipients. MICs found for the donor, recipients, and transconjugants as well as detected resistance genes are given in Table ​Table1.1. Plasmid profiling and hybridization showed that the blaCMY-2 gene was located on a >60-kb plasmid. The pulsed-field gel electrophoresis (PFGE) profile of the isolate was compared to those of 10 other serovar Heidelberg isolates from food products or animals in Denmark and four isolates from humans. PFGE was performed by using XbaI and BlnI as restriction enzymes according to PulseNet protocol, and its results were compared to those of the PulseNet database. No identical isolates were found. TABLE 1. ICs found for and occurrence of resistance genes in Serovar Heidelberg isolated from an imported boar in Denmark and MICs found for recipients used for conjugation and transconjugants Transferable plasmids (60 to 160 kb) with resistance to ampicillin, cefoxitin, ceftiofur, cephalothin, florfenicol, streptomycin, sulfisoxazole, and tetracycline have previously been detected in Salmonella isolates from the animal and human populations in Canada (1) and the United States (4, 9, 10). The blaCMY-2 gene has so far not been reported in serovar Heidelberg from other countries. However, the location on similar plasmids could indicate that the blaCMY-2 gene in the isolates from Canada, Denmark, and the United States is located on the same transferable genetic resistance element. Serovar Heidelberg is one of the most common serovars causing infections in humans in the United States (7). This serovar is not so far of any major importance in Denmark but clearly has the potential to become an important cause of infections in humans. In addition, since the resistance is located on a transferable plasmid, this resistance might easily spread once it is introduced into an animal population. The present observation showed that a Salmonella isolate with multiresistance was imported by Denmark through the market of live animals. International spread of multiresistant clones has previously been observed (3), and there are several potential routes where this might occur. Trading in live animals provides a very efficient way to spread both pathogenic clones and resistance genes. Breeding animals constitute the top of the production pyramid, and from these herds clones and genes might easily spread to all other animals in the production system. Recent studies from the United States indicate that similar plasmids with the blaCMY-2 genes can be isolated from several sources, including cattle and swine, indicating that plasmids and genes have spread. Salmonella isolates with ampC-mediated β-lactam resistance have so far not become common in Europe. However, in 2003 an outbreak of serovar Newport with blaCMY-2, associated with eating horse meat, was detected in France (http://www.eurosurveillance.org/ew/2003/030703.asp#2). The global spread of ampC-mediated resistance in Salmonella and other Enterobacteriaceae is one of the emerging problems that we presently face. Thus, studies determining how this spread occurs are urgently needed.

Antimicrobial Drug Resistance of Salmonella Isolates from Meat and Humans, Denmark

We compared 8,144 Salmonella isolates collected from meat imported to or produced in Denmark, as well as from Danish patients. Isolates from imported meat showed a higher rate of antimicrobial drug resistance, including multidrug resistance, than did isolates from domestic meat. Isolates from humans showed resistance rates lower than those found in imported meat but higher than in domestic meat. These findings indicate that programs for controlling resistant Salmonella spp. are a global issue.

Source attribution of human campylobacteriosis in Denmark

SUMMARY This study assesses the contribution of different sources of human campylobacteriosis in Denmark using two different source-attribution approaches. In total, 794 non-human isolates and 406 isolates from human cases (domestic, travel related, and cases with unknown travel history) were collected. Isolates were characterized by multilocus sequence typing, flaA typing and susceptibility to antibiotics. Both models used indicate that the major burden of human campylobacteriosis in Denmark originates from the domestic broiler chicken reservoir. The second most important reservoir was found to be cattle. The Asymmetric Island model attributed 52% [95% credibility interval (CrI) 37-67] to Danish chicken, 17% (95% CrI 3-33) to imported chicken, and 17% (95% CrI 7-28) to cattle. Similarly, the Campylobacter source-attribution model apportioned 38% (95% CrI 28-47) to Danish chicken, 14% (95% CrI 10-18) to imported chicken, and 16% (95% CrI 7-25) to cattle. The addition of flaA type as an extra discriminatory typing parameter did not change the attribution of cases markedly.

Molecular Characterization of Salmonella Typhimurium Highly Successful Outbreak Strains

Three large clusters of Salmonella Typhimurium infections in Denmark in 2008 and 2009 were defined by multilocus variable number of tandem repeat analysis (MLVA). One of these proved to be the hereto largest Danish cluster of salmonellosis with 1446 cases. Two smaller clusters with a total of 197 and 89 cases, respectively, were seen concurrently. These clusters shared epidemiological characteristics such as age distribution, geography, and time. To investigate the possible genetic relationship between the cluster strains, these were further characterized by phage typing, pulsed-field gel electrophoresis, and Optical Mapping. Although the MLVA method proved robust and well-performing in detecting and defining clusters, the employment of a second typing method detected an additional fourth cluster among the isolates. The cluster strains were stable throughout the almost 2-year period, even though we detected changes in three of five MLVA loci in a small fraction of isolates. These changes were mainly due to the gain or loss of single repeats. Optical Mapping of the large cluster strain indicated no increased content of virulence genes; however, Optical Mapping did reveal a large insert, a probable prophage, in the main cluster. This probable prophage may give the cluster strain a competitive advantage. The molecular methods employed suggested that the four clusters represented four distinct strains, although they seemed to be epidemiologically linked and shared genotypic characteristics.

A regional outbreak of S. Typhimurium in Denmark and identification of the source using MLVA typing.

In Denmark, as part of the national laboratory-based surveillance system of human enteric infections, all S. Typhimurium isolates are currently sub-typed using phage typing, antibiogram typing, and pulsed-field gel electrophoresis (PFGE). However, the discriminatory ability of PFGE is not always high enough to discriminate within certain phage types, and it is not always possible to separate unrelated and related isolates. We have therefore applied multiple locus variable number of tandem repeats analysis (MLVA) for surveillance typing of S. Typhimurium since 2004. In May and June 2005, an outbreak with 26 cases of S. Typhimurium infection was identified by MLVA. The isolates were fully sensitive and had one of the most frequently occurring Danish phage types (DT12) and PFGE types. S. Typhimurium DT12 isolates from routine surveillance of animals and food were typed using MLVA and PFGE for comparison with the human isolates. The typing results revealed that an isolate from a pig herd and its corresponding slaughterhouse located in the same geographic region as the outbreak had the same PFGE and MLVA type as the human isolates. In contrast, all other DT12 isolates investigated, which had the same PFGE profile, had different MLVA types. The conclusion that the pig herd was the source of the human infections was supported by patient information, and pork from the herd stopped entering the market on 29 June. MLVA may contribute significantly to both surveillance and outbreak investigations of S. Typhimurium, as without MLVA typing this outbreak would not have been found nor its origin traced.

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.

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.

A long-lasting outbreak of Salmonella Typhimurium U323 associated with several pork products, Denmark, 2010

This paper shows that control of foodborne disease outbreaks may be challenging even after establishing the source of infection. An outbreak of Salmonella Typhimurium U323 infections occurred in Denmark from March to September 2010, involving 172 cases. Before the detection of human cases, several positive isolates of the outbreak strain had been found in a particular pig slaughterhouse and thus early traceback, investigation and control measures were possible. Several batches of pork and pork products were recalled and the slaughterhouse was closed twice for disinfection. No single common food item was identified as the outbreak source, but repeated isolation of the outbreak strain from the slaughterhouse environment and in pork and products as well as patient interviews strongly suggested different pork products as the source of infection. Furthermore, a matched case-control study identified a specific ready-to-eat spreadable pork sausage (teewurst) as the source of a sub-outbreak (matched odds ratio 17, 95% confidence interval 2·1-130).