Investigation of an international outbreak of multidrug-resistant monophasic Salmonella Typhimurium associated with chocolate products, EU/EEA and United Kingdom, February to April 2022.

An extensive multi-country outbreak of multidrug-resistant monophasic Salmonella Typhimurium infection in 10 countries with 150 reported cases, predominantly affecting young children, has been linked to chocolate products produced by a large multinational company. Extensive withdrawals and recalls of multiple product lines have been undertaken. With Easter approaching, widespread product distribution and the vulnerability of the affected population, early and effective real-time sharing of microbiological and epidemiological information has been of critical importance in effectively managing this serious food-borne incident.

The Epidemiology of Monophasic Salmonella Typhimurium.

Salmonella enterica remains an important foodborne pathogen in all regions of the world, with Typhimurium as one of the most frequent serotypes causing foodborne disease. However, the past two decades have seen a rapid worldwide emergence of a new Salmonella serotype, namely monophasic variant of S. Typhimurium, whose antigenic formula is 1,4,[5],12:i:-. It has become one of the 2-5 most common Salmonella serotypes responsible for animal and human infections in different regions. The global epidemic of monophasic S. 1,4,[5],12:i:- has mainly been characterized by an increase in multidrug-resistant S. 1,4,[5],12:i:- isolated in Europe since 1997. The unexpected link to swine has escalated monophasic S. Typhimurium infections to the status of a global public health emergency. The large-scale application of whole genome sequencing (WGS) in the last 10 years has revealed the phylogenetic associations of the bacterium and its antimicrobial resistance (AMR) genes. Local and global transmission reconstructed by WGS have shown that different clones have emerged following multiple independent events worldwide, and have elucidated the role of this zoonotic pathogen in the spread of AMR. This article discusses our current knowledge of the global ecology, epidemiology, transmission, bacterial adaptation, and evolution of this emerging Salmonella serotype.

Increasing incubation periods during a prolonged monophasic Salmonella Typhimurium outbreak with environmental contamination of a commercial kitchen at Oslo Airport, Norway, 2017.

In September 2017, a cluster of monophasic Salmonella Typhimurium isolates was identified at the National Reference Laboratory for Enteropathogenic Bacteria in Norway. We investigated the cluster to identify the source and implement control measures. We defined a case as a person with laboratory-confirmed salmonellosis with the outbreak strain multiple locus variable-number tandem repeat analysis type. We conducted descriptive epidemiological and environmental investigations and performed whole genome sequencing (WGS) with core and accessory genome multilocus sequence typing of all isolates from cases or the environment connected with this outbreak. We identified 21 cases, residing in 10 geographically dispersed counties, all of whom had consumed food or drinks from a café at Oslo Airport. Case distribution by date of symptom onset suggested that a point source was introduced in mid-August followed by continued environmental contamination. The incubation periods ranged 0-16 days and increased as the outbreak progressed, likely due to increasingly low-dose exposure as control measures were implemented. WGS confirmed an identical cluster type-944 in all cases and six environmental specimens from the café. Control measures, including temporary closure and kitchen refurbishment, failed to eliminate the environmental source. We recommend strengthened hygiene measures for established environmental contamination during an outbreak.

Observations on the introduction and dissemination of Salmonella in three previously low prevalence status pig farms in the United Kingdom.

In the United Kingdom a serological Salmonella surveillance scheme was run in pigs up to 2012. Farms that maintained a low seroprevalence (<10%) were recognised as "Platinum" pig farms. The aim of this study was to investigate the occurrence and distribution of Salmonella in three farms (17P, 18P and 46P) that had lost their "Platinum" status. Four visits to each farm were made over a period of 15 months. The sampling was carried out by collecting pooled pen floor faecal swab and environmental samples. All samples were tested for Salmonella by a modification of ISO6579 Annex D, and serovars were determined for all isolates. The Salmonella prevalence peaked in the Summer/Autumn months and all farms were still positive at the end of the study. The overall sample prevalence was higher in farm 17P (46%) and 18P (35%) than 46P (19%). Monophasic S. Typhimurium (mST) represented 77.8% of the Salmonella isolates, mainly from farms 17P and 46P. The mST isolated at the initial visit may have been introduced via other livestock present on farm or introduction into the herd of infected animals. The results of this study suggest that incursion of mST was likely to be the main cause of the loss of "Platinum" status and confirm that mST can persist in pigs and their environment.

Colonization of Pigs Experimentally Infected with a Monophasic Variant of Salmonella Typhimurium.

The monophasic variant of Salmonella Typhimurium is highly prevalent in human and in pork. However, little is known about colonization dynamics and serology in pigs. We orally inoculated 24 seven-week-old piglets with 109 CFU/pig of a porcine strain of monophasic Salmonella Typhimurium in an experimental trial. Three groups of eight piglets were orally inoculated and monitored for 21, 49, or 84 days post-inoculation until necropsied. From 3 days post-inoculation to necropsy, individual feces were sampled twice weekly and blood once weekly. At necropsy, the tonsils, mesenteric lymph nodes, and the contents of the duodenum, jejunum, ileum, and cecum were collected from each pig. We determined the number of CFU/g in all the samples and measured also Salmonella antibodies in OD% in all blood samples. At different times during the trial, we tested by MLVA (Multilocus Variable Number Tandem Repeat Analysis) the genomic stability of the strain after passing through the intestinal tract. Salmonella was continuously excreted by pigs, ranging from 1.4 to 5.8 log10 CFU/g. At necropsy, Salmonella was present in all samples, but the tonsils were particularly infected. Salmonella antibodies were detected in five pigs 7 days post-inoculation. At 49 days post-inoculation, all the pigs were seropositive. We observed new MLVA types for 3.3% of the isolates tested over the trial. Our study allowed us to show the serovar's ability to persist in pigs after infection up to 84 days post-inoculation. We demonstrated that Salmonella seroconversion appeared earlier than in naturally infected pigs and that the strain's genome can evolve after passing through the digestive tract of pigs.

Evaluation of an Alternative Experimental Infection Method, Which Closely Mimics the Natural Route of Transmission of Monophasic Salmonella Typhimurium in Pigs.

Salmonella carriage in pigs is a significant food safety issue. This study describes a new protocol of Salmonella infection based on exposure to an artificially contaminated environment that closely mimics natural exposure to the organism. The aim of the study was to develop and evaluate the effectiveness of this protocol, which could then be used as a tool in the investigation of control measures. In addition, Salmonella shedding pattern and growth performance of the pigs were examined. Trial pigs (n = 10) were placed in a pen that had been previously contaminated by housing two pigs experimentally challenged with a monophasic Salmonella Typhimurium (mST). A further 10 pigs were placed in a Salmonella-free pen. Pigs were weighed on days 0 and 28. Feces was collected on days 0, 2, 3, 5, 7, 14, 21, and 28 and examined for the presence and quantity of Salmonella. The trial was replicated once. All pigs in the contaminated pens shed Salmonella within the first 2 days of exposure with values ranging from 100 to 104 CFU/g. The noninfected pigs had significantly higher final body weights on day 28 than those exposed to the Salmonella contaminated environment in both replicates. The pigs in the Salmonella-free pen had significantly higher average daily weight gain over the 28-day period compared to the infected animals (p < 0.001). Although not significant, numerical improvements in average daily feed intake and feed conversion efficiency were observed in the Salmonella-free pigs when compared to the contaminated pigs. The approach used was successful in infecting pigs with Salmonella without the need for direct inoculation or exposure to seeder pigs. This "natural" method of infection in which pigs are exposed to low levels of environmental contamination with Salmonella may be an effective tool that could be utilized when investigating control measures.

Eurasian griffon vultures carry widespread antimicrobial resistant Salmonella and Campylobacter of public health concern.

The global emergence of antimicrobial-resistant (AMR) strains of Salmonella and Campylobacter is a serious public health concern. Both bacteria are leading causes of human gastrointestinal foodborne infections and the two most reported zoonoses in the European Union. By feeding on livestock carcasses, especially from intensive farming, as well as on landfill sites, obligate avian scavengers can become infected with zoonotic pathogens and AMR strains, and can be considered large-scale sentinels of the environmental burden. In this study, we assessed the occurrence and AMR of Salmonella spp. and Campylobacter spp. in 218 Eurasian griffon vultures (Gyps fulvus) captured in north-eastern Spain. We isolated Salmonella from 8.1 % of individuals and Campylobacter lari from 4.7 %. Among the 10 different Salmonella serovars found, monophasic S. Typhimurium was the most frequent. Genotyping analysis revealed same strains of monophasic S. Typhimurium shared by gulls, livestock and humans. Isolates from both bacterial species presented AMR to important antimicrobials (tetracyclines, fluoroquinolones and ß-lactams). In conclusion, this study shows that Eurasian griffon vultures in north-eastern Spain are carriers of widespread AMR zoonotic Salmonella and Campylobacter. More comprehensive analyses are still needed to understand the potential risk of spill-over from those wild birds to humans.

Phenotypic and genotypic antibiotic resistance patterns in Salmonella Typhimurium and its monophasic variant from pigs in southern Spain.

The high incidence of human salmonellosis and multi-drug resistant (MDR) strains of Salmonella Typhimurium (ST) is of concern to global public and animal health. Our research, by means of the broth microdilution method, evaluated the Minimum Inhibitory Concentration (MIC) distribution of 12 antimicrobials against a collection of 73 ST and mST and S. typhimurium monophasic variant 4,[5],12:i:- (mST) isolates from slaughtered pigs reared in extensive systems in southern Spain, and also 12 resistance-associated genes or antimicrobial resistance (AMR) determinants using qPCR. Our data revealed that 98.6% of strains were MDR, with resistance to cephalothin/tetracycline/sulfamethoxazole-trimethoprim/ampicillin/chloramphenicol being the most common pattern (55.6%). Regarding AMR determinants, the most significantly (p < 0.05) genes detected by qPCR were sul1 and aadA2 (89% of strains positive), aadA1 and dfrA12 (87.7%), and blaTEM and tet(B) (86.3% and 84.9%, respectively). Up to date information on ST antimicrobial resistance patterns is essential for epidemiological surveillance programs to support animal and public health. The high number of MDR isolates and variability regarding resistance determinants revealed in this study highlights the role of animals reared in extensive systems as a source of resistant Salmonella strains.

Outbreak of monophasic Salmonella Typhimurium Sequence Type 34 linked to chocolate products.

As of 3rd June 2022, 445 cases of monophasic Salmonella Typhimurium sequence type 34 infection had been reported globally. The outbreak was caused by two novel strains of monophasic S. Typhimurium with unusual multi-drug resistance. The majority of these cases involved children aged 10 or younger, and they had a hospitalization rate higher than most previous outbreaks of monophasic S. Typhimurium, but no fatalities were recorded. The infection was traced to certain Belgian chocolate products after extensive microbiological and epidemiological research. Public health officials took immediate action to recall all the contaminated products, and the risk of exposure was reduced. The common symptoms are bloody diarrhea, acute onset of fever, abdominal pain, and vomiting. This article aims to thoroughly review the recent outbreak of monophasic Salmonella Typhimurium ST-34, including its epidemiology and comparison with ongoing outbreaks. We also highlighted past chocolate-related salmonella outbreaks and current control and prevention guidelines and recommendations.

Observations on the distribution and persistence of monophasic Salmonella Typhimurium on infected pig and cattle farms.

Following a rapid rise in cases of monophasic Salmonella Typhimurium DT193 (mST) in humans and pigs since 2007 a detailed study of the prevalence and persistence of mST on pig and cattle farms in Great Britain (GB) was undertaken. Thirteen commercial pig farms and twelve cattle farms, identified as mST-positive from surveillance data, were intensively sampled over a three year period. Five indoor and eight outdoor pig farms and four beef and eight dairy farms were included. Individual and pooled faecal samples were collected from each epidemiological group and environmental samples throughout each farm and the antimicrobial resistance profile determined for a selection of mST-positive isolates. Indoor pig farms had a higher mST prevalence than outdoor pig farms, and across both cattle and pig farms the juvenile animals had a higher mST prevalence than the adult animals. Overall, mST prevalence decreased with time across all pig farms, from 25% to less than 15% of environmental samples and 22% to 15% of pooled faecal samples; only one organic outdoor breeding farm was Salmonella-negative at the end of the study. Across the cattle farms no mST was detected by the end of the study, apart from one persistent farm. Clearance time of mST was between seven and twenty-five months. Farms were selected based on having the antimicrobial resistance profile ampicillin, streptomycin, sulphonamides and tetracycline (A, S, SU, T), although resistance to trimethoprim-potentiated sulphamethoxazole was also identified on five pig farms sampled. This study provided a detailed insight into the distribution and persistence of mST on individual pig and cattle farms in GB. It has identified variation in mST shedding of individual animals, and the data can be applied to the wider livestock industry when considering the distribution of mST once identified on an individual farm.