Challenges Associated with Investigating Salmonella Enteritidis with Low Genomic Diversity in New York State: The Impact of Adjusting Analytical Methods and Correlation with Epidemiological Data.

Defining investigation-worthy genomic clusters among strains of Salmonella Enteritidis is challenging because of their highly clonal nature. We investigated a cluster identified by core genome multilocus sequence typing (cgMLST) consisting of 265 isolates with isolation dates spanning two and a half years. This cluster experienced chaining, growing to a range of 14 alleles. The volume of isolates and broad allele range of this cluster made it difficult to ascertain whether it represented a common-source outbreak. We explored laboratory-based methods to subdivide and refine this cluster. These methods included using cgMLST with a narrower allele range, whole genome multilocus sequence typing (wgMLST) and high-quality single-nucleotide polymorphism (hqSNP) analysis. At each analysis level, epidemiologists retroactively reviewed exposures, geography, and temporality for potential commonalities. Lowering the threshold to 0 alleles using cgMLST proved an effective method to refine this analysis, resulting in this large cluster being subdivided into 34 smaller clusters. Additional analysis by wgMLST and hqSNP provided enhanced cluster resolution, with the majority of clusters being further refined. These analysis methods combined with more stringent allele thresholds and layering of epidemiologic data proved useful in helping to subdivide this large cluster into actionable subclusters.

Prevalence and antimicrobial resistance of Salmonella enterica subspecies enterica serovar Enteritidis isolated from broiler chickens in Shandong Province, China, 2013-2018.

Salmonella is a major zoonotic foodborne pathogen that persists on poultry farms worldwide. The present study aimed to survey the prevalence of Salmonella and antimicrobial resistance of Salmonella enterica serovar Enteritidis (S. Enteritidis) recovered from broiler chickens in Shandong Province, China. A total of 280 Salmonella isolates were identified from 923 broiler chicken samples between 2013 and 2018. Among the isolates, S. Enteritidis (n = 128, 45.7%) was the predominant serovar, and high antimicrobial resistance rates to piperacillin (PIP) (n = 123, 96.1%), ampicillin (AM) (n = 122, 95.3%), nitrofurantoin (FT) (n = 106, 96.1%), and tetracycline (TE) (n = 93, 72.7%) were observed in S. Enteritidis. A total of 96 (75.0%) S. Enteritidis isolates presented with multidrug resistance, the most frequent of which were the combination of AM, PIP, TE, and FT. Resistance to fluoroquinolone tended to increase during 2013 to 2018. Our findings provide important and updated information about the baseline antimicrobial-resistant data for food safety and a risk assessment of S. Enteritidis from broiler chickens in Shandong Province and will be helpful for future surveillance activities to ensure the safety of the chicken supply.

Comparison of conventional molecular and whole-genome sequencing methods for subtyping Salmonella enterica serovar Enteritidis strains from Tunisia.

We sought to determine the relative value of conventional molecular methods and whole-genome sequencing (WGS) for subtyping Salmonella enterica serovar Enteritidis recovered from 2000 to 2015 in Tunisia and to investigate the genetic diversity of this serotype. A total of 175 Salmonella Enteritidis isolates were recovered from human, animal, and foodborne outbreak samples. Pulsed-field gel electrophoresis (PFGE), multiple locus variable-number tandem repeat analysis (MLVA), and whole-genome sequencing were performed. Eight pulsotypes were detected for all isolates with PFGE (DI = 0.518). Forty-five Salmonella Enteritidis isolates were selected for the MLVA and WGS techniques. Eighteen MLVA profiles were identified and classified into two major clusters (DI = 0.889). Core genome multilocus typing (cgMLST) analysis revealed 16 profiles (DI = 0.785). Whole-genome analysis indicated 660 single-nucleotide polymorphism (SNP) divergences dividing these isolates into 43 haplotypes (DI = 0.997). The phylogenetic tree supported the classification of Salmonella Enteritidis isolates into two distinct lineages subdivided into five clades and seven subclades. Pairwise SNP differences between the isolates ranged between 302 and 350. We observed about 311 SNP differences between the two foodborne outbreaks, while only less or equal to 4 SNP differences within each outbreak. SNP-based WGS typing showed an excellent discriminatory power comparing with the conventional methods such as PFGE and MLVA. Besides, we demonstrate the added value of WGS as a complementary subtyping method to discriminate outbreak from non-outbreak isolates belonging to common subtypes. It is important to continue the survey of Salmonella Enteritidis lineages in Tunisia using WGS.

Phylogenetic structure of Salmonella Enteritidis provides context for a foodborne outbreak in Peru.

Salmonella Enteritidis, an important foodborne zoonosis, has a dramatically increased number of cases around the world. To explore the phylogenetic structure of Peruvian Salmonella Enteritidis strains and their relationship with an outbreak occurred in 2018, we analyzed a comprehensive strains of S. Enteritidis received by the National Institute of Health during the period 2000-2018. A total of 180 strains were characterized by microbiological procedures, serotyping and whole genome sequencing. Based on genome sequences annotated, virulence factors and accessory genes were identified. Phylogenetic and population structure analysis were also analyzed based on SNPs. The phylogenetic analysis grouped the genomes into two well-supported clades that were consistent with population structure analysis. The clinical and food strains corresponding to the outbreak were included in the same cluster, which presented the sdhA gene, related to the increase of the virulence of this pathogen. The phylogenetic relationship of Peruvian S. Enteritidis suggests the presence of four S. enteritidis population with high epidemiological importance.

Analysis of Salmonella enterica serovar Enteritidis isolates from chickens and chicken meat products in Malaysia using PFGE, and MLST.

BACKGROUND: Salmonella is a very important foodborne pathogen causing illness in humans. The emergence of drug-resistant strains also constitutes a serious worry to global health and livestock productivity. This study investigated Salmonella isolates from chicken and chicken meat products using the phenotypic antimicrobial screening as well as the molecular characteristics of Salmonella isolates. Upon serotyping of the isolates, the antimicrobial susceptibility profiling using a panel of 9 commonly used antimicrobials was done. Subsequently, the molecular profiles of all the isolates were further determined using Pulsed Field Gel Electrophoresis (PFGE) and the Whole Genome Multi-Locus Sequence Type (wgMLST) analysis in order to obtain the sequence types. RESULTS: The PFGE data was input into FPQuest software, and the dendrogram generated was studied for possible genetic relatedness among the isolates. All the isolates were found to belong to the Salmonella Enteritidis serotype with notable resistance to tetracycline, gentamycin, streptomycin, and sulfadimidine. The S. Enteritidis isolates tested predominantly subtyped into the ST11 and ST1925, which was found to be a single cell variant of ST11. The STs were found to occur in chicken meats, foods, and live chicken cloacal swabs, which may indicate the persistence of the bacteria in multiple foci. CONCLUSION: The data demonstrate the presence of S. Enteritidis among chickens, indicating its preference and reservoir status for enteric Salmonella pathogens.

Highly clonal relationship among Salmonella Enteritidis isolates in a commercial chicken production chain, Brazil.

In this study, we described the comparison among pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD), ribotyping, and PCR-ribotyping methods for subtyping Salmonella Enteritidis isolated from an industrial chicken production chain. One hundred and eight S. Enteritidis were isolated at all stages of poultry meat processing plant. These isolates were pheno- and genotypically characterized by using antimicrobial susceptibility test, phage typing, RAPD, PFGE, ribotyping, and PCR-ribotyping. The highest antibiotic resistance rates were observed for enrofloxacin (18.5%) followed by furazolidone (15.7%), cefoxitin (1.8%), ciprofloxacin, and ampicillin with 0.9% each one, while seven isolates (6.4%) were pan-susceptible. Most strains belonged to the globally disseminated phage type PT4 (n = 74; 69.2%). Additionally, we identified strains belonging to phage types PT1 (n = 19; 17.8%) and PT7a (n = 14; 13.1%). Moreover, our results showed that these four molecular methods indicate similar results showing high similarity (≥ 90%) among S. Enteritidis strains, suggesting that these isolates appear to be from a common ancestor being spread at all stages of the poultry production chain. In summary, the combined molecular approaches of these methods remain a suitable alternative to efficiently subtyping S. Enteritidis in the absence of high-resolution genotyping methods and these results may serve as a baseline study for development of mitigation strategies.

Prediction of Salmonella serovars isolated from clinical and food matrices in Lebanon and genomic-based investigation focusing on Enteritidis serovar.

Salmonella enterica subsp. enterica serovars are considered major causes of food poisoning and we performed this study because Salmonella is a burden in Lebanon. The present study investigated the ability of genomic information to predict serovar using a collection of Salmonella isolates from infected humans (n = 24) and contaminated food (n = 63) in Lebanon. Further, the phylogenomic relationships of the serovar the predominated in Lebanon (i.e., S. Enteritidis; n = 25) were investigated in comparison with isolates from other countries (n = 130) based on coregenome single nucleotide polymorphisms (SNPs). Genetic elements, specifically Salmonella pathogenicity islands (SPIs), plasmid replicons, and antibiotic-resistance genes were screened in S. Enteritidis genomes (n = 155). Our results revealed that the Salmonella serovars identification by seroagglutination from the samples isolated in Lebanon (n = 87) was highly correlated with the genomic-based prediction of serovars (80.4-85.0% with SeqSero1 and 93.1-94.2% with SeqSero2). The Salmonella serovars isolated from human and food samples in Lebanon were mainly Enteritidis (28.7%) and Infantis (26%). To a rare extent, other serovars included Amager, Anatum, Bredeney, Chincol, Heidelberg, Hofit, Kentucky, Montevideo, Muenster, Newport, Schwarzengrund, Senftenberg and Typhimurium. In comparison with other countries, S. Enteritidis samples isolated in Lebanon (56 ± 27 intra-group pairwise SNP differences) presented a strong phylogenomic relativeness at the coregenome level with samples, as for example with samples isolated from Syria (65 ± 31 inter-group pairwise SNP differences). Most of the studied S. Enteritidis genomes encoded 10 SPIs involved in survival in immune cells (i.e. SPIs 1, 2, 3, 4, 5, 12, 13, 14, 16 and 17). The plasmid replicons IncFIB (S)_1 and IncFII (S)_1 encoding elements involved in virulence were identified in the majority of the S. Enteritidis genomes (94% and 96%, respectively), the majority exhibiting aminoglycosides (gene aac(6')-Iaa_1). The IncI_1_Alpha replicon responsible for ampicillin-resistance was only detected in 2 of 25 S. Enteritidis Lebanese strains. Genomic-based risk assessment of Salmonella serovars in Lebanon showed that food imported from Syria might be an origin of the S. Enteritidis human cases in Lebanon. The detection of several SPIs involved in the survival, plasmid replicons involved in virulence, and aminoglycoside-resistance genes, emphasizes that S. Enteritidis is of paramount importance for public health in Lebanon and other countries.

Genomic analyses of multidrug-resistant Salmonella Indiana, Typhimurium, and Enteritidis isolates using MinION and MiSeq sequencing technologies.

We sequenced 25 isolates of phenotypically multidrug-resistant Salmonella Indiana (n = 11), Typhimurium (n = 8), and Enteritidis (n = 6) using both MinION long-read [SQK-LSK109 and flow cell (R9.4.1)] and MiSeq short-read (Nextera XT and MiSeq Reagent Kit v2) sequencing technologies to determine the advantages of each approach in terms of the characteristics of genome structure, antimicrobial resistance (AMR), virulence potential, whole-genome phylogeny, and pan-genome. The MinION reads were base-called in real-time using MinKnow 3.4.8 integrated with Guppy 3.0.7. The long-read-only assembly, Illumina-only assembly, and hybrid assembly pipelines of Unicycler 0.4.8 were used to generate the MinION, MiSeq, and hybrid assemblies, respectively. The MinION assemblies were highly contiguous compared to the MiSeq assemblies but lacked accuracy, a deficiency that was mitigated by adding the MiSeq short reads through the Unicycler hybrid assembly which corrected erroneous single nucleotide polymorphisms (SNPs). The MinION assemblies provided similar predictions of AMR and virulence potential compared to the MiSeq and hybrid assemblies, although they produced more total false negatives of AMR genotypes, primarily due to failure in identifying tetracycline resistance genes in 11 of the 19 MinION assemblies of tetracycline-resistant isolates. The MinION assemblies displayed a large genetic distance from their corresponding MiSeq and hybrid assemblies on the whole-genome phylogenetic tree, indicating that the lower read accuracy of MinION sequencing caused incorrect clustering. The pan-genome of the MinION assemblies contained significantly more accessory genes and less core genes compared to the MiSeq and hybrid assemblies, suggesting that although these assemblies were more contiguous, their sequencing errors reduced accurate genome annotations. Our research demonstrates that MinION sequencing by itself provides an efficient assessment of the genome structure, antimicrobial resistance, and virulence potential of Salmonella; however, it is not sufficient for whole-genome phylogenetic and pan-genome analyses. MinION in combination with MiSeq facilitated the most accurate genomic analyses.

Genome sequence analysis of 91 Salmonella Enteritidis isolates from mice caught on poultry farms in the mid 1990s.

A total of 91 draft genome sequences were used to analyze isolates of Salmonella enterica serovar Enteritidis obtained from feral mice caught on poultry farms in Pennsylvania. One objective was to find mutations disrupting open reading frames (ORFs) and another was to determine if ORF-disruptive mutations were present in isolates obtained from other sources. A total of 83 mice were obtained between 1995-1998. Isolates separated into two genomic clades and 12 subgroups due to 742 mutations. Nineteen ORF-disruptive mutations were found, and in addition, bigA had exceptional heterogeneity requiring additional evaluation. The TRAMS algorithm detected only 6 ORF disruptions. The sefD mutation was the most frequently encountered mutation and it was prevalent in human, poultry, environmental and mouse isolates. These results confirm previous assessments of the mouse as a rich source of Salmonella enterica serovar Enteritidis that varies in genotype and phenotype.

Genome sequence of a clinical Salmonella Enteritidis sequence type 11 strain from South Africa.

OBJECTIVES: The underlying resistance mechanism and phylogenetic relationship of a colistin-resistant Salmonella enterica serovar Enteritidis strain EC20120916 that resulted in fatal meningitis in an immunocompromised patient was investigated by whole-genome sequencing (WGS) analysis. METHODS: WGS of strain EC20120916 was performed on an Illumina MiSeq platform and annotation of the sequence was performed using the Prokaryotic Genome Annotation Pipeline (PGAP). Antimicrobial resistance genes, plasmid replicons and pathogenicity islands were identified. A phylogenetic tree was constructed using Parsnp and was edited with FigTree. RESULTS: The genome size of strain EC20120916 is 4 699 318 bp with a GC content of 55.2% and 4471 protein-coding genes. The aac(6')-laa gene, encoding resistance to aminoglycosides, was identified although this was not expressed phenotypically in the isolate. No colistin resistance-conferring mutations or plasmid-mediated mechanisms were identified to explain the colistin resistance. The strain was phylogenetically related to three international strains, although it was not close enough to suggest importation from outside of South Africa. CONCLUSION: This is the first report of a colistin-resistant Salmonella Enteritidis isolate causing meningitis in an immunocompromised patient in South Africa. The absence of colistin resistance-conferring mutations or plasmid-mediated resistance mechanisms suggest that a novel mechanism is responsible for the colistin resistance in this isolate. The isolate was acquired locally.

A common Salmonella Enteritidis sequence type from poultry and human gastroenteritis in Ibagué, Colombia / Un tipo de secuencia común de Salmonella Enteritidis de origen aviar y de humano con gastroenteritis en Ibagué, Colombia

Salmonella Enteritidis es una de las mayores causas de salmonelosis en el mundo, siendo los huevos contaminados y la carne de pollo cruda sus principales fuentes de infección. En Ibagué, Colombia, se identificaron los principales serovares circulando en granjas, superficies de huevos y canales de pollo, sin embargo, se desconoce si esos serovares son responsables de gastroenteritis. Objetivo. Evaluar la relación genética entre aislamientos de Salmonella Enteritidis de aves de corral y humanos con gastroenteritis mediante multilocus sequence typing (MLST). Materiales y métodos. Se aisló Salmonella spp., de casos clínicos de gastroenteritis (n=110). Se realizó test de sensibilidad antibiótica, seguido de serotipificación y tipificación por medio de MLST y se comparó S. Enteritidis de humanos frente a S. Enteritidis de granjas ponedoras y de huevo comercializado (n=6). Resultados. Se aislaron 10 cepas de Salmonella spp., a partir de heces de humanos con gastroenteritis. Se obtuvo una prevalencia de Salmonella spp. de 9.09%, siendo S. Enteritidis (n=4), S. Typhymurium (n=2), S. Newport (n=1), S. Grupensis (n=1), S. Uganda (n=1) y S. Braenderup (n=1) los serotipos presentes en pacientes con gastroenteritis. El MLST indico que un tipo de secuencia común (ST11) de S. Enteritidis estuvo presente en todas las tres fuentes y mostraron el mismo patrón de resistencia antibiótica. Conclusión. S. Enteritidis ST11 constituye un vínculo entre el consumo/manipulación de huevos contaminados y gastroenteritis humana en Ibagué. Son necesarios estudios complementarios para conocer si otros serovares de Salmonella aislados de carne de pollo cruda también se asocian con la gastroenteritis humana.

Introducción. Salmonella Enteritidis es una de las mayores causas de salmonelosis en el mundo, siendo los huevos contaminados y la carne de pollo cruda sus principales fuentes de infección. En Ibagué, Colombia, se identificaron los principales serovares circulando en granjas, superficies de huevos y canales de pollo, sin embargo, se desconoce si esos serovares son responsables de gastroenteritis. Objetivo. Evaluar la relación genética entre aislamientos de Salmonella Enteritidis de aves de corral y humanos con gastroenteritis mediante multilocus sequence typing (MLST). Materiales y métodos. Se aisló Salmonella spp., de casos clínicos de gastroenteritis (n=110). Se realizó test de sensibilidad antibiótica, seguido de serotipificación y tipificación por medio de MLST y se comparó S. Enteritidis de humanos frente a S. Enteritidis de granjas ponedoras y de huevo comercializado (n=6). Resultados. Se aislaron 10 cepas de Salmonella spp., a partir de heces de humanos con gastroenteritis. Se obtuvo una prevalencia de Salmonella spp. de 9.09%, siendo S. Enteritidis (n=4), S. Typhymurium (n=2), S. Newport (n=1), S. Grupensis (n=1), S. Uganda (n=1) y S. Braenderup (n=1) los serotipos presentes en pacientes con gastroenteritis. El MLST indico que un tipo de secuencia común (ST11) de S. Enteritidis estuvo presente en todas las tres fuentes y mostraron el mismo patrón de resistencia antibiótica. Conclusión. S. Enteritidis ST11 constituye un vínculo entre el consumo/manipulación de huevos contaminados y gastroenteritis humana en Ibagué. Son necesarios estudios complementarios para conocer si otros serovares de Salmonella aislados de carne de pollo cruda también se asocian con la gastroenteritis humana.

S. Enteritidis and S. Typhimurium Harboring SPI-1 and SPI-2 Are the Predominant Serotypes Associated With Human Salmonellosis in Saudi Arabia.

Non-typhoidal Salmonella (NTS) strains are Gram negative bacterial pathogens that are associated with foodborne illness worldwide. During the process of infection, Salmonella uses two molecular injectisomes known as Type 3 Secretion Systems (T3SS) to secrete virulence factors that are encoded by Salmonella Pathogenicity Island-1 (SPI-1) and SPI-2 into host cells. These secretion systems play a major role in virulence, as shown in various animal models, but little is known about their role in human infections. In Saudi Arabia, NTS strains frequently cause human infections but data regarding these pathogenic strains is fairly limited. The aim of this study was to characterize Salmonella human clinical isolates in Riyadh, Saudi Arabia, by determining their serotype, testing for the presence of SPI-1 and SPI-2 genes and to determine the antibiotic resistance profiles of these strains. Using the rapid Check and Trace Salmonella™ (CTS) system our results demonstrate that S. Enteritidis and S. Typhimurium were the predominant serovars, followed by S. Livingstone, S. Kentucky and S. Poona among a list of 36 serovars reported for the first time in the country. In addition, SPI-1 genes were detected in 99% of the isolates, while the sifA gene (SPI-2) was not detected in 13.5% of the isolates. These results suggest that both the SPI-1 and SPI-2 virulence determinants are important for human infection. Moreover, we report the presence of a Multi-Drug (MDR) carbapenem resistant S. Kentucky isolate harboring the blaOXA-48 gene not reported previously in Saudi Arabia.

Emergence of phylogenetically diverse and fluoroquinolone resistant Salmonella Enteritidis as a cause of invasive nontyphoidal Salmonella disease in Ghana.

BACKGROUND: Salmonella enterica serovar Enteritidis is a cause of both poultry- and egg-associated enterocolitis globally and bloodstream-invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa (sSA). Distinct, multi-drug resistant genotypes associated with iNTS disease in sSA have recently been described, often requiring treatment with fluoroquinolone antibiotics. In industrialised countries, antimicrobial use in poultry production has led to frequent fluoroquinolone resistance amongst globally prevalent enterocolitis-associated lineages. METHODOLOGY/PRINCIPAL FINDINGS: Twenty seven S. Enteritidis isolates from patients with iNTS disease and two poultry isolates, collected between 2007 and 2015 in the Ashanti region of Ghana, were whole-genome sequenced. These isolates, notable for a high rate of diminished ciprofloxacin susceptibility (DCS), were placed in the phyletic context of 1,067 sequences from the Public Health England (PHE) S. Enteritidis genome database to understand whether DCS was associated with African or globally-circulating clades of S. Enteritidis. Analysis showed four of the major S. Enteritidis clades were represented, two global and two African. All thirteen DCS isolates, containing a single gyrA mutation at codon 87, belonged to a global PT4-like clade responsible for epidemics of poultry-associated enterocolitis. Apart from two DCS isolates, which clustered with PHE isolates associated with travel to Spain and Brazil, the remaining DCS isolates, including one poultry isolate, belonged to two monophyletic clusters in which gyrA 87 mutations appear to have developed within the region. CONCLUSIONS/SIGNIFICANCE: Extensive phylogenetic diversity is evident amongst iNTS disease-associated S. Enteritidis in Ghana. Antimicrobial resistance profiles differed by clade, highlighting the challenges of devising empirical sepsis guidelines. The detection of fluoroquinolone resistance in phyletically-related poultry and human isolates is of major concern and surveillance and control measures within the region's burgeoning poultry industry are required to protect a human population at high risk of iNTS disease.

A common Salmonella Enteritidis sequence type from poultry and human gastroenteritis in Ibagué, Colombia / Un tipo de secuencia común de Salmonella Enteritidis de origen aviar y de humano con gastroenteritis en Ibagué, Colombia

Abstract Introduction: Salmonella Enteritidis is a major cause of human salmonellosis in the world, with contaminated eggs and raw chicken meat as the main routes of infection. The main Salmonella spp. serovars circulating in laying hen farms, the surface of eggs, and in raw chicken carcasses have been identified in Ibagué, Colombia. However, it is unknown whether those serovars are responsible for human gastroenteritis. Objective: To evaluate the genetic relationship between gastroenteritis and Salmonella Enteritidis isolates from poultry and humans using multilocus sequence typing (MLST). Materials and methods: Salmonella spp. was isolated from clinical cases of gastroenteritis (n=110). Antibiotic susceptibility tests, followed by serotyping and MLST were conducted and S. Enteritidis was compared to those from laying hen farms and marketed eggs. Results: Ten isolates of Salmonella spp. were obtained from the stools of people with gastroenteritis. The prevalence of Salmonella spp. in human stools was 9.09%, and S. Enteritidis (n=4), S. Typhymurium (n=2), S. Newport (n=1), S. Uganda (n=1), S. Grupensis (n=1), and S. Braenderup (n=1) were the main serotypes. MLST indicated that a common S. Enteritidis sequence type (ST11) was present in all three sources and showed the same antibiotic resistance pattern. Conclusion: Salmonella Enteritidis ST11 constitutes a link between consumption and manipulation of contaminated eggs and human gastroenteritis in Ibagué. Additional studies would be required to establish if other Salmonella serovars isolated from raw chicken meat are also associated with human gastroenteritis.

Resumen Introducción. Salmonella Enteritidis es una de las mayores causas de salmonelosis en el mundo; los huevos contaminados y la carne de pollo cruda son sus principales fuentes de infección. En Ibagué, Colombia, se han identificado los principales serovares que circulan en granjas, superficies de huevos y canales de pollo, pero se desconoce si esos serovares son responsables de la gastroenteritis. Objetivo. Evaluar la relación genética entre los aislamientos de Salmonella Enteritidis de aves de corral y de humanos con la gastroenteritis mediante tipificación de multiloci de secuencias (Multilocus Sequence Typing, MLST). Materiales y métodos. Se aisló Salmonella spp. de casos clínicos de gastroenteritis (n=110). Se hizo la prueba de sensibilidad antibiótica, así como la serotipificación y la tipificación mediante MLST, y se comparó S. Enteritidis de humanos con la hallada en granjas de gallinas ponedoras y en huevo comercializado (n=6). Resultados. Se aislaron 10 cepas de Salmonella spp. a partir de heces de humanos con gastroenteritis. Se obtuvo una prevalencia de Salmonella spp. de 9,09%, y se identificaron los serotipos S. Enteritidis (n=4), S. Typhymurium (n=2), S. Newport (n=1), S. Grupensis (n=1), S. Uganda (n=1) y S. Braenderup presentes en pacientes con gastroenteritis. Mediante la MLST, se comprobó que un tipo de secuencia común (ST11) de S. Enteritidis estuvo presente en todas las tres fuentes y presentó el mismo patrón de resistencia antibiótica. Conclusión. Salmonella Enteritidis ST11 constituye un vínculo entre el consumo y la manipulación de huevos contaminados, y la gastroenteritis en humanos en Ibagué. Se requieren estudios complementarios para conocer si otros serovares de Salmonella aislados de carne de pollo cruda también se asocian con la gastroenteritis en humanos.

Discrimination of non-typhoid Salmonella serogroups and serotypes by Fourier Transform Infrared Spectroscopy: A comprehensive analysis.

Simpler, quick and low-cost methods for routine Salmonella enterica typing are required for epidemiologic surveillance of this important zoonotic pathogen. In this study, using a comprehensive isolate collection, we investigated the potential of Fourier transform infrared spectroscopy (FTIRS) to discriminate the most clinically-relevant serogroups and serotypes of non-typhoid Salmonella. Moreover, the role of O-units composition on the FTIRS Salmonella discrimination was also explored. S. enterica isolates (n = 325; 2002-2015; different sources and countries), of 57 serotypes and 15 serogroups [including the most frequent ones, B-n = 122; C-n = 108; D-n = 43 and E-n = 33)] were analysed by FTIRS. Infrared spectra were analysed by Partial Least Square Discriminant Analysis (PLSDA) and/or Principal Component Analysis (PCA). The polysaccharides region provided the spectral sharpest differences being used in the subsequent Salmonella typing. Serogroups (B, C, D and E) discrimination was achieved with high accuracy (99.6% of correct assignments; PLSDA model). Differences in the O-unit structures composition of those serogroups are likely justifying the discrimination achieved. Other serogroups (G, H, K, L, M, N, O, T, U, Y, Z) were correctly predicted as not belonging to serogroups B, C, D nor E, except for 3 isolates of serogroups H (S. Sundsvall, n = 1) and K (S. Cerro, n = 2). In fact, O-unit structure of serogroup H and K shows some similarity with sub-serogroup C1 with the remaining serogroups presenting marked differences in this cellular component. The sub-serogroups discrimination was successfully achieved for C1, C2 and C3 (using PCA), and for E1-E2-E3 and E4 (by PLSDA). Appropriate serotype discrimination was obtained for most of S. Rissen from the remaining C1 serotypes (91.5%-PLSDA), and S. Enteritidis (D1) from the remaining D1/D2 serotypes (93.4%-PLSDA). The lack of available O-unit composition for particular serotypes prevents the elucidation of the role of this cellular component on the discrimination at serotype level obtained. FTIRS was able to discriminate relevant serogroups (B, C, D and E), sub-serogroups (C1, C2 and C3; E1-E2-E3 and E4) and particular important serotypes (S. Enteritidis, S. Rissen and S. Senftenberg). Further studies on O-antigen composition would clarify the fundaments of discrimination obtained by FTIRS.

Retrospective genome-wide comparisons of Salmonella enterica serovar Enteritidis from suspected outbreaks in Singapore.

The number of salmonellosis cases in Singapore has increased over the years. Salmonella enterica serovar Enteritidis has always been the most predominant serovar in the last five years. The National Public Health Laboratory assisted outbreak investigations by performing multilocus variable number tandem repeat analysis (MLVA) on isolates that were collected at the time of the investigations. Isolates were defined as belonging to a particular cluster if they had identical MLVA patterns. Whilst MLVA has been instrumental in outbreak investigations, it may not be useful when outbreaks are caused by an endemic MLVA type. In this study, we analysed 67 isolates from 12 suspected outbreaks with known epidemiological links to explore the use of next-generation sequencing (NGS) for defining outbreaks. We found that NGS can confidently group isolates into their respective outbreaks. The isolates from each suspected outbreak were closely related and differed by a maximum of 3 single nucleotide polymorphisms (SNPs). They were also clearly separated from isolates that belonged to different suspected outbreaks. This study provides an important insight and further evidence on the value of NGS for routine surveillance and outbreak detection of S. Enteritidis.

Susceptibility Distribution to Essential Oils of Salmonella enterica Strains Involved in Animal and Public Health and Comparison of the Typhimurium and Enteritidis Serotypes.

To determine the distribution of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of essential oils (EOs) of cinnamon (Cinnamomum zeylanicum), clove (Eugenia caryophyllata), oregano (Origanum vulgare), common thyme (Thymus vulgaris), and red thyme (Thymus zygis) against Salmonella enterica, double serial dilutions of each EO were challenged with 85 Salmonella strains belonging to 23 serotypes of animal origin. The results showed the bactericidal character of the EOs tested against S. enterica, highlighting the oregano with MIC50 and MBC50 of 3.12 × 10-4 g/mL, and MIC90 and MBC90 of 6.25 × 10-4 g/mL. When comparing the Salmonella Typhimurium and Salmonella Enteritidis serotypes susceptibility, we observed a significantly higher sensitivity of Typhimurium to clove and Enteritidis to cinnamon. In addition, Typhimurium isolates with significantly higher MIC and MBC values for all the EOs tested were found, suggesting the existence of a possible resistance profile. The results of this study provide relevant data for the potential of EOs as antibacterials, although they highlight the need to continue bacterial sensitivity distribution studies and consider the differences detected for future in vivo studies.

Rapid molecular identification and differentiation of common Salmonella serovars isolated from poultry, domestic animals and foodstuff using multiplex PCR assay.

Salmonella is widely distributed throughout the world and can be found in poultry industry, animal breeding centers, food and feedstuffs of all geographical regions. This study was conducted to determine and identify Salmonella serovars isolated from poultry, calves and foodstuffs (poultry and animals products such as egg and meat). A total of one hundred isolates of Salmonella serovars including Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Infantis, Salmonella Gallinarum and Salmonella Pullorum consecutively were subjected to the conventional culture, biochemical and serological assays. The utility of molecular multiplex PCR was investigated to identify and differentiate among five Salmonella serovars which were identified according to the presence of rfbJ, fljB, invA, and fliC genes in S. Typhimurium, sefA, invA and spv genes in Salmonella Enteritidis, fljB, fliC and invA genes in Salmonella Infantis, hut and slgC genes in both Salmonella Gallinarum and Salmonella Pullorum and speC gene specifically in Salmonella Gallinarum. Biochemical assays and serotyping are complicated to directly differentiate between Salmonella Gallinarum and Salmonella Pullorum because of their antigenic similarity. According to the results, Multiplex PCR can be considered as simple, rapid, accurate and useful test to identify and differentiate among Salmonella serovars.

Salmonella Enteritidis ST183: emerging and endemic biotypes affecting western European hedgehogs (Erinaceus europaeus) and people in Great Britain.

The impacts of hedgehog (Erinaceus europaeus) Salmonella infection on public health and on animal welfare and conservation are unknown. We isolated Salmonella Enteritidis multi-locus sequence-type (ST)183 from 46/170 (27%) hedgehog carcasses (27 S. Enteritidis phage type (PT)11, 18 of a novel PT66 biotype and one with co-infection of these PTs) and from 6/208 (3%) hedgehog faecal samples (4 PT11, 2 PT66) from across Great Britain, 2012-2015. Whole genome phylogenetic analysis of the hedgehog isolates and ST183 from people in England and Wales found that PT11 and PT66 form two divergent clades. Hedgehog and human isolates were interspersed throughout the phylogeny indicating that infections in both species originate from a common population. PT11 was recovered from hedgehogs across England and Scotland, consistent with endemic infection. PT66 was isolated from Scotland only, possibly indicating a recent emergence event. People infected with ST183 were four times more likely to be aged 0-4 years than people infected by the more common ST11 S. Enteritidis. Evidence for human ST183 infection being non-foodborne included stronger correlation between geographic and genetic distance, and significantly increased likelihood of infection in rural areas, than for ST11. These results are consistent with hedgehogs acting as a source of zoonotic infection.

Whole genome sequencing reveals an outbreak of Salmonella Enteritidis associated with reptile feeder mice in the United Kingdom, 2012-2015.

Analysis of whole genome sequencing data uncovered a previously undetected outbreak of Salmonella Enteritidis that had been on-going for four years. Cases were resident in all countries of the United Kingdom and 40% of the cases were aged less than 11 years old. Initial investigations revealed that 30% of cases reported exposure to pet snakes. A case-control study was designed to test the hypothesis that exposure to reptiles or their feed were risk factors. A robust case-definition, based on the single nucleotide polymorphism (SNP) profile, increased the power of the analytical study. Following univariable and multivariable analysis, exposure to snakes was the only variable independently associated with infection (Odds ratio 810 95% CI (85-7715) p < 0.001). Isolates of S. Enteritidis belonging to the outbreak profile were recovered from reptile feeder mice sampled at the retail and wholesale level. Control measures included improved public health messaging at point of sale, press releases and engagement with public health and veterinary counterparts across Europe. Mice destined to be fed to reptiles are not regarded as pet food and are not routinely tested for pathogenic bacteria. Routine microbiological testing to ensure feeder mice are free from Salmonella is recommended.