The Salmonella PathogeniCity Island (SPI) 1 contributes more than SPI2 to the colonization of the chicken by Salmonella enterica serovar Typhimurium

Background  

Salmonella enterica serovar Typhimurium (Typhimurium) is an important pathogen that infects a broad range of hosts. In humans, Typhimurium causes a gastroenteritis characterized by vomiting, diarrhea, and abdominal pains. Typhimurium infection occurs mainly through the ingestion of contaminated food including poultry, pork, eggs, and milk. Chickens that are asymptomatic carriers of Typhimurium constitute a potential reservoir for infection. The type three secretion systems encoded by Salmonella pathogeniCity islands (SPI) 1 and 2 are major virulence factors of Salmonella. However, only a few studies have investigated their role during the infection of chickens.     

Preslaughter Holding Environment in Pork Plants Is Highly Contaminated with Salmonella enterica

The objective of this study was to determine whether abattoir pens can provide a Salmonella enterica infection source during the 2 to 4 h of preharvest holding. Previous work has suggested that pigs may be getting infected, but little has been reported on the environmental contamination of abattoir holding pens. For 24 groups of pigs studied (~150 animals/group) at two high-capacity abattoirs, six pooled fecal samples (n, 10 per pool) were collected from each transport trailer immediately after pigs were unloaded. Holding pens were sampled (one drinking water sample and six pooled floor samples consisting of swabs, residual liquid, and feces) prior to entry of study pigs for the routine holding period (~2.5 h). After slaughter, cecal contents and ileocecal lymph nodes were collected, on the processing line, from 30 pigs in each studied group. All samples were cultured for the isolation and identification of S. enterica by primary enrichment in GN-Hajna and tetrathionate broths, secondary enrichment in Rappaport-Vassiliadis broth, and plating on brilliant green sulfa and xylose-lysine-tergitol-4 agars, followed by biochemical and serological identification. The study pens were highly contaminated with S. enterica; all holding pens sampled had at least one positive sample. Additionally, 33% (8 of 24) of drinking water samples were positive for S. enterica. All 24 groups of pigs had S. enterica-positive cecal contents and ileocecal lymph nodes, including those groups from transport trailers with no positive samples. From pigs, trailers, and pens, 586 isolates representing 36 different Salmonella serovars were isolated. Of the 353 isolates from pigs (109 from ileocecal lymph nodes plus 244 from cecal contents), 19% were identified as belonging to the same serovars as those isolated from the respective pens; 27% were identified as belonging to the same serovars as those isolated from the trailers. Sixteen percent of the unique serovars were isolated from both pigs and pens, suggesting that pens served as the infection source. This study demonstrates highly contaminated abattoir holding pens and watering sources. It also demonstrates that holding pens can serve as an infection source. This study identifies the abattoir holding pens as a significant hazard and a potential control point for Salmonella contamination in the preharvest pork production chain.     

Multi‐locus sequence typing of Salmonella enterica serovar Typhimurium isolates from wild birds in northern England suggests host‐adapted strain

Aims: Recent studies have suggested that Salmonella Typhimurium strains associated with mortality in UK garden birds are significantly different from strains that cause disease in humans and livestock and that wild bird strains may be host adapted. However, without further genomic characterization of these strains, it is not possible to determine whether they are host adapted. The aim of this study was to characterize a representative sample of Salm. Typhimurium strains detected in wild garden birds using multi‐locus sequence typing (MLST) to investigate evolutionary relationships between them. Methods and Results: Multi‐locus sequence typing was performed on nine Salm. Typhimurium strains isolated from wild garden birds. Two sequence types were identified, the most common of which was ST568. Examination of the public Salmonella enterica MLST database revealed that only three other ST568 isolates had been cultured from a human in Scotland. Two further isolates of Salm. Typhimurium were determined to be ST19. Conclusions: Results of MLST analysis suggest that there is a predominant strain of Salm. Typhimurium circulating among garden bird populations in the United Kingdom, which is rarely detected in other species, supporting the hypothesis that this strain is host adapted. Significance and Impact of the Study: Host–pathogen evolution is often assumed to lead to pathogens becoming less virulent to avoid the death of their host; however, infection with ST568 led to high mortality rates among the wild birds examined, which were all found dead at wild bird‐feeding stations. We hypothesize that by attracting unnaturally high densities of birds, wild bird‐feeding stations may facilitate the transmission of ST568 between wild birds, therefore reducing the evolutionary cost of this pathogen killing its host, resulting in a host‐adapted strain with increased virulence.     

Salmonella enterica Infections in Market Swine with and without Transport and Holding

The objective of this study was to compare, by using identical sample types, the Salmonella enterica prevalences and serovar diversities between pigs necropsied on the farm and those necropsied at the abattoir after transport and holding. We necropsied 567 market weight pigs (>70 kg) from six herds. Pigs were alternately assigned to be necropsied on the farm or at the abattoir. One-half of the group was sent in clean, disinfected trailers to slaughter at a commercial abattoir. After transport (mean distance, 169 km) and 2 to 3 h of holding in antemortem pens, these pigs were necropsied. The 50 pigs remaining on the farm were necropsied the following day. The same sample types and amounts were collected for S. enterica culture at both locations. Results show a sevenfold-higher (P < 0.001) S. enterica isolation rate from pigs necropsied at the abattoir (39.9%; 114 of 286) than from those necropsied on the farm (5.3%; 15 of 281). This difference was also observed for each individual herd. All sample types showed a significantly higher prevalence when comparing abattoir to on-farm collection, respectively: lymph nodes, 9.15 versus 3.6%; cecal contents, 13.6 versus 1.8%; 1 g of fecal matter, 25.2 versus 0.7%. Recovery of additional serovars at the abattoir suggests the pigs are receiving S. enterica from extra-farm sources. This study demonstrates that rapid infection during transport, and particularly during holding, is a major reason for increased S. enterica prevalence in swine. This finding identifies the holding pen as an important S. enterica control point in the pork production chain.     

Salmonella enterica infections in market swine with and without transport and holding

The objective of this study was to compare, by using identical sample types, the Salmonella enterica prevalences and serovar diversities between pigs necropsied on the farm and those necropsied at the abattoir after transport and holding. We necropsied 567 market weight pigs (>70 kg) from six herds. Pigs were alternately assigned to be necropsied on the farm or at the abattoir. One-half of the group was sent in clean, disinfected trailers to slaughter at a commercial abattoir. After transport (mean distance, 169 km) and 2 to 3 h of holding in antemortem pens, these pigs were necropsied. The 50 pigs remaining on the farm were necropsied the following day. The same sample types and amounts were collected for S. enterica culture at both locations. Results show a sevenfold-higher (P < 0.001) S. enterica isolation rate from pigs necropsied at the abattoir (39.9%; 114 of 286) than from those necropsied on the farm (5.3%; 15 of 281). This difference was also observed for each individual herd. All sample types showed a significantly higher prevalence when comparing abattoir to on-farm collection, respectively: lymph nodes, 9.15 versus 3.6%; cecal contents, 13.6 versus 1.8%; 1 g of fecal matter, 25.2 versus 0.7%. Recovery of additional serovars at the abattoir suggests the pigs are receiving S. enterica from extra-farm sources. This study demonstrates that rapid infection during transport, and particularly during holding, is a major reason for increased S. enterica prevalence in swine. This finding identifies the holding pen as an important S. enterica control point in the pork production chain.     

Phenotypic and Molecular Typing of Salmonella Strains Reveals Different Contamination Sources in Two Commercial Pig Slaughterhouses

This study aimed to define the origin of Salmonella contamination on swine carcasses and the distribution of Salmonella serotypes in two commercial slaughterhouses during normal activity. Salmonellae were isolated from carcasses, from colons and mesenteric lymph nodes of individual pigs, and from the slaughterhouse environment. All strains were serotyped; Salmonella enterica serotype Typhimurium and Salmonella enterica serotype Derby isolates were additionally typed beyond the serotype level by pulsed-field gel electrophoresis (PFGE) and antibiotic resistance profiling (ARP); and a subset of 31 serotype Typhimurium strains were additionally phage typed. PFGE and ARP had the same discriminative possibility. Phage typing in combination with PFGE could give extra information for some strains. In one slaughterhouse, 21% of the carcasses were contaminated, reflecting a correlation with the delivery of infected pigs. Carcass contamination did not result only from infection of the corresponding pig; only 25% of the positive carcasses were contaminated with the same serotype or genotype found in the corresponding feces or mesenteric lymph nodes. In the other slaughterhouse, 70% of the carcasses were contaminated, and only in 4% was the same genotype or serotype detected as in the feces of the corresponding pigs. The other positive carcasses in both slaughterhouses were contaminated by genotypes present in the feces or lymph nodes of pigs slaughtered earlier that day or from dispersed sources in the environment. In slaughterhouses, complex contamination cycles may be present, resulting in the isolation of many different genotypes circulating in the environment due to the supply of positive animals and in the contamination of carcasses, probably through aerosols.     

Case report of clinical salmonellosis by Salmonella Typhimurium that occurred in Portuguese children

Aims: Aim of this study is to characterize clinical isolates of Salmonella Typhimurium that occurred in Portuguese children on the basis of their virulence and antimicrobial resistance profiles and pulsed‐field gel electrophoresis typing and to analyse possible strain relatedness. Methods and Results: Different Salmonella serotypes were isolated from clinical cases of salmonellosis that had occurred in two Portuguese hospitals (a total of 259 isolates). All Salm. Typhimurium strains, with the age of the patients known, (total of 26 isolates) were selected for this study. These isolates were characterized for their virulence gene profiles (agfA, iroB, slyA, hin/H2, spv), antimicrobial resistance profiles and investigated for the occurrence of multidrug‐resistant Salm. Typhimurium DT 104 by PCR. Salmonella isolates showed high rates of resistance to four or more antibiotics, 100% resistance to sulfadiazine and a high percentage of strains with the resistance profile of Salm. Typhimurium DT 104, two of them with this phage type (determined by PCR). A relationship between some clusters and their resistance and virulence profiles was detected, each cluster having the same profile. Conclusions: This study showed high‐antibiotic resistance of the Salmonella strains investigated, and the presence of multidrug‐resistant Salm. Typhimurium DT104 in infections of Portuguese children. Significance and Impact of the Study: Study is based on regarding the increase in antibiotic resistance by Salmonella strains isolated from infections in Portuguese children and on the presence of Salm. Typhimurium DT 104 circulating in Portugal.     

Identification by PCR of Non-typhoidal Salmonella enterica Serovars Associated with Invasive Infections among Febrile Patients in Mali

Background

In sub-Saharan Africa, non-typhoidal Salmonella (NTS) are emerging as a prominent cause of invasive disease (bacteremia and focal infections such as meningitis) in infants and young children. Importantly, including data from Mali, three serovars, Salmonella enterica serovar Typhimurium, Salmonella Enteritidis and Salmonella Dublin, account for the majority of non-typhoidal Salmonella isolated from these patients.

Methods

We have extended a previously developed series of polymerase chain reactions (PCRs) based on O serogrouping and H typing to identify Salmonella Typhimurium and variants (mostly I 4,[5],12:i:-), Salmonella Enteritidis and Salmonella Dublin. We also designed primers to detect Salmonella Stanleyville, a serovar found in West Africa. Another PCR was used to differentiate diphasic Salmonella Typhimurium and monophasic Salmonella Typhimurium from other O serogroup B, H:i serovars. We used these PCRs to blind-test 327 Salmonella serogroup B and D isolates that were obtained from the blood cultures of febrile patients in Bamako, Mali.

Principal Findings

We have shown that when used in conjunction with our previously described O-serogrouping PCR, our PCRs are 100% sensitive and specific in identifying Salmonella Typhimurium and variants, Salmonella Enteritidis, Salmonella Dublin and Salmonella Stanleyville. When we attempted to differentiate 171 Salmonella Typhimurium (I 4,[ 5],12:i:1,2) strains from 52 monophasic Salmonella Typhimurium (I 4,[5],12:i:-) strains, we were able to correctly identify 170 of the Salmonella Typhimurium and 51 of the Salmonella I 4,[5],12:i:- strains.

Conclusion

We have described a simple yet effective PCR method to support surveillance of the incidence of invasive disease caused by NTS in developing countries.     

Dose Determination for Acute Salmonella Infection in Pigs

Pigs were exposed to various levels of Salmonella enterica subsp. enterica serovar Typhimurium by either intranasal inoculation or by subjecting them to a contaminated environment. More than 10³ salmonellae were required to induce acute Salmonella infection. These results indicate that intervention against acute Salmonella infection in lairage may be more readily achieved than previously thought.     

Different mutations in the oafA gene lead to loss of O5-antigen expression in Salmonella enterica serovar Typhimurium

Aims: To analyse genetic changes in the oafA gene explaining the loss of O5‐antigen expression in Salmonella Typhimurium and Salm. 4,[5],12:i:‐. Methods and Results: The oafA gene in 52 O5‐antigen‐negative and 77 O5‐antigen‐positive Salm. Typhimurium (N = 47) and Salm. 4,[5],12:i:‐ (monophasic Salm. Typhimurium strains, N = 82) was investigated by a combination of PCR screening and DNA sequencing to identify mutations leading to the suppression of the O5‐antigen. Various DNA sequence changes within the open reading frame (ORF) of oafA in O5‐antigen‐negative strains could be identified. In 77% of the O5‐antigen‐negative strains, a 7‐bp deletion of a duplicated sequence within the functional oafA gene led to a frameshift in the ORF. In four strains, an IS4 element and in two, an IS1 element was inserted at different positions. Four other strains carried at different positions single base pair substitutions causing a premature stop codon. Finally, in two strains, a deletion of the oafA 3′end of undetermined size was responsible for the lack of O5‐antigen expression. In none of the strains investigated, the complete ORF of oafA was deleted. Primers were designed and used to detect the most prominent variants. Conclusions: O5‐antigen‐negative Salm. Typhimurium and Salm. 4,[5],12:i:‐ strains carry an oafA pseudogene caused by different genetic events indicating that there is a selection for oafA mutations leading to the loss of O5‐antigen expression. Significance and Impact of the Study: The loss of O5‐antigen expression may be an example of a common evolutionary mechanism to escape host defence or to adapt to environmental changes. The data are the basis for the development of diagnostic PCR assays for the differentiation of O5‐antigen‐positive and O5‐antigen‐negative Salm. Typhimurium and its monophasic (Salm. 4,[5],12:i‐) strains.     

Characterization of the RpoS Status of Clinical Isolates of Salmonella enterica

The stationary-phase-inducible sigma factor, σ^S (RpoS), is the master regulator of the general stress response in Salmonella and is required for virulence in mice. rpoS mutants can frequently be isolated from highly passaged laboratory strains of Salmonella. We examined the rpoS status of 116 human clinical isolates of Salmonella, including 41 Salmonella enterica serotype Typhi strains isolated from blood, 38 S. enterica serotype Typhimurium strains isolated from blood, and 37 Salmonella serotype Typhimurium strains isolated from feces. We examined the abilities of these strains to produce the σ^S protein, to express RpoS-dependent catalase activity, and to resist to oxidative stress in the stationary phase of growth. We also carried out complementation experiments with a cloned wild-type rpoS gene. Our results showed that 15 of the 41 Salmonella serotype Typhi isolates were defective in RpoS. We sequenced the rpoS allele of 12 strains. This led to identification of small insertions, deletions, and point mutations resulting in premature stop codons or affecting regions 1 and 2 of σ^S, showing that the rpoS mutations are not clonal. Thus, mutant rpoS alleles can be found in freshly isolated clinical strains of Salmonella serotype Typhi, and they may affect virulence properties. Interestingly however, no rpoS mutants were found among the 75 Salmonella serotype Typhimurium isolates. Strains that differed in catalase activity and resistance to hydrogen peroxide were found, but the differences were not linked to the rpoS status. This suggests that Salmonella serotype Typhimurium rpoS mutants are counterselected because rpoS plays a role in the pathogenesis of Salmonella serotype Typhimurium in humans or in the transmission cycle of the disease.     

SulA-induced filamentation in Salmonella enterica serovar Typhimurium: effects on SPI-1 expression and epithelial infection

Aims: Salmonella enterica serovar Typhimurium is capable of adopting a filamentous phenotype in response to damage. How this adaptive response affects bacterial virulence is unclear. We have examined the hypothesis that filamentation affects the ability of Salmonella to infect host cells. Methods and Results: Expression of the cell division inhibitor SulA in Salm. Typhimurium SL1344 from an arabinose‐inducible plasmid resulted in filamentation. We examined expression of the type 3 secretion system (T3SS) encoded by Salmonella pathogenicity island 1 (SPI‐1) using SL1344 expressing a chromosomal PprgH‐gfp reporter. Single cell analysis of SulA‐induced SL1344 PprgH‐gfp revealed a relationship between increasing cell length and decreasing propensity for prgH expression, but there was no evidence of a significant change in prgH expression evident at the whole population level. Filamentous Salm. Typhimurium were capable of initiating membrane ruffling on MDCK epithelial cells, but only nonfilamentous bacteria (<6 μm) invade. Conclusions: Induction of SulA expression in Salmonella inhibits septation. Increasing filament length is associated with down‐regulation of SPI‐1 gene expression, but a significant proportion of filaments retain the ability to produce SPI‐1 T3SS and induce membrane ruffles on epithelia. Despite an active SPI‐1 T3SS, filamentous Salmonella are unable to invade epithelial cells. Significance and Impact of the Study: Our findings that filamentous Salmonella can express an invasive phenotype but fail to invade cells suggest that their presence in food does not constitute an immediate risk of infection until septation occurs. The described SulA expression model provides a convenient model for studying the impact of filamentation in the absence of additional stresses.     

Resistance to essential oils affects survival of Salmonella enterica serovars in growing and harvested basil

The number of outbreaks of food‐borne illness associated with consumption of fresh products has increased. A recent and noteworthy outbreak occurred in 2007. Basil contaminated with Salmonella enterica serovar Senftenberg was the source of this outbreak. Since basil produces high levels of antibacterial compounds the aim of this study was to investigate if the emerging outbreak reflects ecological changes that occurred as a result of development of resistance to ingredients of the basil oil. We irrigated basil plants with contaminated water containing two Salmonella serovars, Typhimurium and Senftenberg, and showed that Salmonella can survive on the basil plants for at least 100 days. S. Senftenberg counts in the phyllosphere were significantly higher than S. Typhimurium, moreover, S. Senftenberg was able to grow on stored harvested basil leaves. Susceptibility experiments demonstrated that S. Senftenberg is more resistant to basil oil and to its antimicrobial constituents: linalool, estragole and eugenol. This may indicate that S. Senftenberg had adapted to the basil environment by developing resistance to the basil oil. The emergence of resistant pathogens has a significant potential to change the ecology, and opens the way for pathogens to survive in new niches in the environment such as basil and other plants.     

The protective effect of Bdellovibrio‐and‐like organisms (BALO) on tilapia fish fillets against Salmonella enterica ssp. enterica serovar Typhimurium

Aims: To characterize freshwater Bdellovibrio‐and‐like organisms (BALO) isolated in China and examine their potential in controlling growth of Salmonella enterica ssp. enterica serovar Typhimurium on tilapia fillets. Methods and Results: Four BALO isolates were recovered from a pond in Yanzhou of Shandong province, China, with Salm. Typhimurium as prey using double‐layer agar method. Partial 16S rDNA sequencing analysis identified BD2GL, BD5GL and BDXGL as Bdellovibrio bacteriovorus and BD2GS as a Peredibacter sp. Lysis experiments on 32 potentially pathogenic strains revealed that BALO lysis rates are in the range of 56·3–65·6%. On the five Salmonella strains tested, only BD2GS achieved 100% lysis rate. When applied on tilapia fillets against Salm. Typhimurium, BD2GS showed its growth control potential. Cell increments of Salm. Typhimurium were significantly lower (P < 0·05) in two BD2GS‐treated groups compared to control and low‐dose group (BD2GS to prey ratio, 1 : 1) was more effective than high‐dose group (BD2GS to prey ratio, 10 : 1) in controlling Salm. Typhimurium growth. Conclusions: Results of this study indicated that BD2GS could control Salm. Typhimurium growth on tilapia fillets. Significance and Impact of the Study: BALO could be used as a live protective culture in controlling bacterial growth and ensure food safety.     

Use of multiple‐locus variable‐number tandem‐repeats analysis (MLVA) typing to characterize Salmonella Typhimurium DT41 broiler breeder infections

Aims: To characterize isolates of Salmonella Typhimurium DT41 obtained from infected flocks of broiler breeders by multiple‐locus variable‐number tandem‐repeats analysis (MLVA) and compare results with a diverse strain collection from Germany and United Kingdom and isolates from Danish patients. Methods and Results: A total of 102 isolates of Salm. Typhimurium phage type DT41 were MLVA typed. MLVA typing showed 4, 12, 25, 9 and 8 different alleles at the five MLVA loci 9, 5, 6, 10 and 3, respectively. A dendrogram based on MLVA types was constructed, and one large group, nine minor groups and 29 more unrelated MLVA types were obtained. The major group included 20 of the 30 human isolates. Isolates obtained from broiler breeders demonstrated major diversity, indicating the existence of several independent introductions of DT41 at farm level. When comparison was made to isolates included from Germany and England, DT41 seems to be ubiquitous in the wild fauna which might represent a risk factor for poultry. Conclusions: Transmission from Danish broilers to humans was not demonstrated, neither was the transmission from rearing farms to broiler breeder farms. Sources of infection at broiler breeder farm level remained unidentified. Significance and Impact of the Study: Major diversity was demonstrated for DT41 MLVA types. A persisting problem with infection of broiler breeder flocks with DT41 was not reflected in broiler flocks originating from these flocks.     

Enhancement of Immunohistochemical Detection of Salmonella in Tissues of Experimentally Infected Pigs

Salmonella Typhimurium is one of the main pathogens compromising porcine and human health as well as food safety, because it is a prevailing source of foodborne infections due to contaminated pork. A prominent problem in the management of this bacteriosis is the number of subclinically infected carrier pigs. As very little is known concerning the mechanisms allowing Salmonella to persist in pigs, the objective of this study was to develop an immunohistochemical approach for the detection of salmonellae in tissue of pigs experimentally infected with Salmonella Typhimurium. Samples were obtained from a challenge trial in which piglets of the German Landrace were intragastrically infected with Salmonella enterica serovar Typhimurium DT104 (1.4-2.1×10¹⁰ CFU). Piglets were sacrificed on days 2 and 28 post infection. Tissue samples of jejunum, ileum, colon, ileocecal mesenteric lymph nodes (Lnn. ileocolici), and tonsils (Tonsilla veli palatini) were fixed in Zamboni’s fixative and paraffin-embedded. Different immunohistochemical staining protocols were evaluated. Salmonella was detected in varying amounts in the tissues. Brown iron-containing pigments in the lymph nodes interfered with the identification of Salmonella if DAB was used as a staining reagent. Detergents like Triton X-100 or Saponin enhanced the sensitivity. It seems advisable not to use a detection system with brown staining for bacteria in an experimental setup involving intestinal damage including haemorrhage. The use of detergents appears to result in a higher sensitivity in the immunohistochemical detection of salmonellae.Key words: Swine, immunohistochemistry, histochemistry, bacteria, detection limit     

Maternal vaccination as a Salmonella Typhimurium reduction strategy on pig farms

Aims

The control of Salmonella in pig production is necessary for public and animal health, and vaccination was evaluated as a strategy to decrease pig prevalence.

Methods and Results

The study examined the efficacy of a live Salmonella Typhimurium vaccine, administered to sows on eight commercial farrow‐to‐finish herds experiencing clinical salmonellosis or Salmonella carriage associated with S. Typhimurium or its monophasic variants. Results of longitudinal Salmonella sampling were compared against eight similarly selected and studied control farms. At the last visit (~14 months after the start of vaccination), when all finishing stock had been born to vaccinated sows, both faecal shedding and environmental prevalence of Salmonella substantially declined on the majority of vaccinated farms in comparison to the controls. A higher proportion of vaccine farms resolved clinical salmonellosis than controls. However, Salmonella counts in positive faeces samples were similar between nonvaccinated and vaccinated herds.

Conclusions

The results suggest that maternal vaccination is a suitable option for a Salmonella Typhimurium reduction strategy in farrow‐to‐finish pig herds.

Significance and Impact of the Study

Salmonella vaccines have the potential to reduce the prevalence of Salmonella in pigs and result in a reduction of human cases attributed to pork.     

Modelling transfer of Salmonella Typhimurium DT104 during simulation of grinding of pork

Aims: The aim of this study was to develop a model to predict cross‐contamination of Salmonella during grinding of pork. Methods and Results: Transfer rates of Salmonella were measured in three experiments, where between 10 and 20 kg meat was ground into 200‐g portions. In each experiment, five pork slices of about 200 g per slice were inoculated with 8–9 log‐units of Salmonella Typhimurium DT104 and used for building up the contamination in the grinder. Subsequently, Salmonella‐free slices were ground and collected as samples of c. 200 g minced pork. Throughout the process, representative samples were quantitatively analysed for Salmonella. A model suggested by Nauta et al. (2005) predicting cross‐contamination of Campylobacter in poultry processing and two modified versions of this model were tested. Conclusions: The present study observed a tailing phenomenon of transfer of Salmonella during a small‐scale grinding process. It was, therefore, hypothesized that transfer occurred from two environmental matrices inside the grinder and a model was developed. The developed model satisfactorily predicted the observed concentrations of Salmonella during its cross‐contamination in the grinding of up to 110 pork slices. Significance and Impact of the Study: The proposed model provides an important tool to examine the effect of cross‐contamination in quantitative microbial risk assessments and might also be applied to various other food processes where cross‐contamination is involved.     

Extensive Genetic Variability Linked to IS26 Insertions in the fljB Promoter Region of Atypical Monophasic Variants of Salmonella enterica Serovar Typhimurium

Fifty-nine monophasic Salmonella enterica serovar Typhimurium isolates, collected in Belgium during the period from 2008 to 2011, have been serotyped as 4,[5]:i:− and shown to harbor an fljB coding sequence. The genetic differences between these strains and phenotypically biphasic Salmonella Typhimurium were analyzed through PCR and DNA sequencing. Genetic alterations in the fljB promoter region affecting expression of the phase 2 flagellin were observed in 53 isolates. Other genetic events in the invertible region carrying the fljB promoter were observed in 2 isolates. For the remaining 4 isolates, no molecular differences with a reference biphasic Salmonella Typhimurium strain could be observed. Next-generation sequencing of one representative isolate affected in the fljB promoter region revealed a 26-kb IS26 composite transposon insertion along with a local genomic rearrangement. Several other IS26 element-mediated alterations of this genomic region were observed. This group of monophasic Salmonella Typhimurium isolates was genetically heterogeneous, as revealed by multilocus variable-number tandem-repeat analysis (MLVA), PCR, and sequencing. Pigs and pork represented a major source of such monophasic isolates in Belgium, as reported in other countries. Three out of 5 isolates of human origin presented genetic profiles identical to those of food isolates, demonstrating the pathogenic potential of the newly characterized variants and potential dissemination along the food chain. This study highlighted the key role played by IS26 insertions in the loss of phase 2 flagellin expression and the subsequent generation of multiple monophasic variant lineages from biphasic Salmonella Typhimurium ancestors.     

A Five-Strain Probiotic Combination Reduces Pathogen Shedding and Alleviates Disease Signs in Pigs Challenged with Salmonella enterica Serovar Typhimurium

Salmonella spp. infection is a major cause of gastroenteritis, with many thousands of cases reported in the European Union every year. The use of probiotics offers the potential to improve this situation. Here, we investigate the effects of oral treatment of pigs with a defined lactic acid bacteria culture mixture on both clinical and microbiological signs of Salmonella enterica serovar Typhimurium infection. Fifteen weaned pigs blocked by sex and weight were administered control milk or a mixture of five probiotic strains as either a milk fermentate or milk suspension for a total of 30 days. The mixture consisted of two strains of Lactobacillus murinus and one strain each of Lactobacillus salivarius subsp. salivarius, Lactobacillus pentosus, and Pediococcus pentosaceous. Following probiotic administration for 6 days, animals were challenged orally with serovar Typhimurium; the health of the animals and the microbiological composition of their feces were monitored for 23 days postinfection. Animals treated with probiotic showed reduced incidence, severity, and duration of diarrhea. These animals also gained weight at a greater rate than control pigs administered skim milk. Mean fecal numbers of Salmonella were significantly reduced in probiotic-treated animals at 15 days postinfection (P = 0.01). The administered probiotic bacteria improved both the clinical and microbiological outcome of Salmonella infection. These strains offer significant benefit for use in the food industry and may have potential in human applications.