Direct or DNA Extraction-Free Amplification and Quantification of Foodborne Pathogens.

The use of direct nucleic acid amplification of pathogens from food matrices has the potential to reduce time to results over DNA extraction-based approaches as well as traditional culture-based approaches. Here we describe protocols for assay design and experiments for direct amplification of foodborne pathogens in food sample matrices using loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). The examples provided include the detection of Escherichia coli in milk samples and Salmonella in pork meat samples. This protocol includes relevant reagents and methods including obtaining target sequences, assay design, sample processing, and amplification. These methods, though used for specific example matrices, could be applied to many other foodborne pathogens and sample types.

The Use of Multiplex Real-Time PCR for the Simultaneous Detection of Foodborne Bacterial Pathogens.

Foodborne pathogens continue to be a major health concern worldwide. Culture-dependent methodologies are still considered the gold standard to perform pathogen detection and quantification. These methods present several drawbacks, such as being time-consuming and labor intensive. The implementation of real-time PCR has allowed to overcome these limitations, and even reduce the cost associated with the analyses, due to the possibility of simultaneously and accurately detecting several pathogens in one single assay, with results comparable to those obtained by classical approaches. In this chapter, a protocol for the simultaneous detection of two of the most important foodborne pathogens, Salmonella spp. and Listeria monocytogenes, is described.

ELIME-IMS hybrid assay for Salmonella detection in swine mesenteric lymph nodes at slaughterhouse.

Salmonella contamination in pig slaughterhouses is linked to infection rate on farms. Accurate diagnosis in heavy pigs relies on isolating pathogens from the gut wall or lymph nodes. A key technique is Immunocapture using Magnetic Beads (IMS), which purifies target bacteria from Salmonella enrichment broths. This is followed by an Enzyme-Linked Immunomagnetic Electrochemical (ELIME) assay for rapid detection. In our study, we developed an ELIME-IMS hybrid assay to detect Salmonella in swine mesenteric lymph nodes (MNL), involving a clean-up with N-acetylcysteine and centrifugation. Detection limits for S. Typhimurium and S. Derby were estimated at 2.80 and 3.52 Log CFU/ml, respectively. We analysed 103 MNL samples from a northern Italy slaughterhouse. Additionally, we examined 15 carcass swabs. Both the ELIME assay and the IMS-based culture method showed strong agreement with the ISO 6579-1:2017 method, especially after 20 h of enrichment (89.47% concordance). The clean-up step significantly influenced the results, as samples processed without it showed higher variability. A logistic regression model indicated high classification accuracy for negative samples using ELIME values. The ELIME-IMS assay facilitates rapid Salmonella screening and isolation in swine mesenteric lymph nodes.

Salmonella in black pepper (Piper nigrum): From farm to processing.

Contamination of black pepper (Piper nigrum) with Salmonella is a frequent problem in retail and imported shipments. However, there is scarce information about the prevalence of the pathogen in the initial stages of black pepper production chain. This study sought to bridge this gap in research by determining the prevalence, as well as quantifying, and identifying the main Salmonella serovars present during black pepper primary production and processing. Black pepper (233) and environmental (175) samples were collected from farms (354) and processing plants (54) in Espirito Santo, Brazil. The pathogen was detected in soil (16.7 %), drying waste (20.4 %), fallen berries (3.7 %), threshed berries (14.3 %), and dried peppercorns (22.2 %) collected from farms. Salmonella was also detected in samples of raw material (11.1 %), export products (16.7 %), and processing waste (16.7 %) collected from processing plants. A total of 12 serotypes were identified, and Salmonella Javiana showed the highest prevalence (38.8 %). According to the results, contamination occurring in the post-harvest phase is not eliminated or reduced during processing. Therefore, the adoption of good agricultural and manufacturing practices, supported by hazard analysis and critical control points (HACCP), is crucial to mitigate this kind of contamination. These practices should be combined with decontamination treatments to ensure the safety of the final product.

Comparative analyses of bacterial contamination and microbiome of broiler carcasses in wet market and industrial processing environments.

The slaughtering environment is crucial for the food hygiene and safety of poultry products. Despite the global dominance of industrial processing, live bird slaughtering in wet markets persists due to cultural, religious, and economic reasons. This study aims to reveal the correlation between hygiene scales in wet markets and bacterial contamination levels on broiler carcasses, with a particular focus on pathobiont transmission risks and microbiome characteristics. Wet markets were categorized based on home-made ratings and the Hygiene and Biosecurity Assessment Tool (HABT). The study assessed total aerobic bacterial (TAB) levels, food spoilage and hygiene indicators (Pseudomonas and E. coli), foodborne pathogen Salmonella, and the microbiome of broiler carcasses, intestinal contents, and slaughtering facilities. Comparative analyses were conducted between market and industrial processing environments. TAB levels on broiler carcasses showed a significant negative correlation with hygiene scores, indicating that both HABT and home-made rating tools effectively assess and improve processing hygiene. Industrial processing consistently reduced bacterial contamination compared to wet markets. Although Salmonella spp. prevalence was lower in market-processed carcasses, the study identified significant cross-transmission of pathobionts and variations in bacterial composition with hygiene improvements. Notably, the microbiome analysis revealed overlaps in amplicon sequence variants (ASVs) between carcasses and contamination vectors, highlighting pathobiont transmission risks. The present study confirmed the scales of hygiene standards among wet markets reflect bacterial contamination on broiler carcasses. Enhancing slaughter practices to meet industrial hygiene standards is essential for reducing the transmission of foodborne pathogens and pathobionts, and improving food safety.

Long-term genomic surveillance reveals the circulation of clinically significant Salmonella in lymph nodes and beef trimmings from slaughter cattle from a Mexican feedlot.

This longitudinal study characterized Salmonella circulating in lymph nodes (LN, n = 800) and beef trimmings (n = 745) from slaughter cattle from a Mexican feedlot. During two years, LN and beef trimming samples were collected 72-96 h post-slaughter, and we obtained 77 isolates of the serovars Anatum (n = 23), Reading (n = 22), Typhimurium (n = 10), London (n = 9), Kentucky (n = 6), Fresno (n = 4), Give, Muenster, and monophasic 1,4,[5],12:i- (n = 1 each). These isolates were subjected to whole genome sequencing, single-nucleotide polymorphism (SNP)-based phylogenetic analysis, reconstruction of their ancestral isolation sources through evolutionary analysis, and virulence profiling. Although LN and beef trimmings were not mixed, evolutionary analysis estimated that the common ancestor of all study isolates was likely of LN origin. Moreover, isolates from both sources were highly clonal (0-21 SNP distance), highlighting the complexity of Salmonella transmission dynamics. The pathogen persisted across cattle cohorts, as shown by clonality between isolates collected in different years (1-20 SNP distance). Major virulence genes were highly conserved (97-100% identity to the reference sequences) and most isolates carried a conserved version of pathogenicity islands 1-5, 9, 11, and 12. Typhimurium strains carried the Salmonella plasmid virulence operon (spvRABCD), and a Muenster isolate carried the st313td gene, both of which are associated with invasive phenotypes. Most isolates (49/77) were genetically similar (1-43 SNPs) to strains involved in human salmonellosis, highlighting their public health significance. Further research is needed on Salmonella transmission dynamics in cattle and the mechanisms determining subclinical infection and persistence in farm environments.

Molecular characterization of common zoonotic protozoan parasites and bacteria causing diarrhea in dairy calves in Ningxia Hui Autonomous Region, China.

Diarrhea caused by zoonotic pathogens is one of the most common diseases in dairy calves, threatening the health of young animals. Humans are also at risk, in particular children. To explore the pathogens causing diarrhea in dairy calves, the present study applied PCR-based sequencing tools to investigate the occurrence and molecular characteristics of three parasites (Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi) and three bacterial pathogens (Escherichia coli, Clostridium perfringens, and Salmonella spp.) in 343 fecal samples of diarrheic dairy calves from five farms in Lingwu County, Ningxia Hui Autonomous Region, China. The total positive rate of these pathogens in diarrheic dairy calves was 91.0% (312/343; 95% CI, 87.9-94.0), with C. perfringens (61.5%, 211/343; 95% CI, 56.3-66.7) being the dominant one. Co-infection with two to five pathogens was found in 67.3% (231/343; 95% CI, 62.4-72.3) of investigated samples. There were significant differences (p < 0.05) in the positive rates of Cryptosporidium spp. and diarrheagenic E. coli among farms, age groups, and seasons. Two Cryptosporidium species (C. parvum and C. bovis) and five gp60 subtypes of C. parvum (IIdA15G1, IIdA20G1, IIdA19G1, IIdA14G1, and a novel IIdA13G1) were identified. Two assemblages (assemblage E and zoonotic assemblage A) of G. duodenalis and six ITS genotypes of E. bieneusi (J, Henan-IV, EbpC, I, EbpA, and ESH-01) were observed. Four virulence genes (eaeA, stx1, stx2, and st) of diarrheagenic E. coli and one toxin type (type A) of C. perfringens were detected. Our study enriches our knowledge on the characteristics and zoonotic potential of diarrhea-related pathogens in dairy calves.

Title: Caractérisation moléculaire des protozoaires parasites zoonotiques courants et des bactéries responsables de diarrhée chez les veaux laitiers dans la région autonome Hui du Ningxia, en Chine. Abstract: La diarrhée causée par des agents pathogènes zoonotiques est l'une des maladies les plus courantes chez les veaux laitiers, menaçant la santé des jeunes animaux. Ceci est également un risque pour la santé humaine, en particulier les enfants. Pour explorer les agents pathogènes responsables de la diarrhée chez les veaux laitiers, cette étude a utilisé des outils de séquençage basés sur la PCR pour étudier l'occurrence et les caractères moléculaires de trois parasites (Cryptosporidium spp., Giardia duodenalis et Enterocytozoon bieneusi) et de trois agents pathogènes bactériens (Escherichia coli, Clostridium perfringens et Salmonella spp.) dans 343 échantillons fécaux de veaux laitiers diarrhéiques provenant de cinq fermes du comté de Lingwu, région autonome Hui du Ningxia, en Chine. Le taux total positif de ces pathogènes chez les veaux laitiers diarrhéiques était de 91,0 % (312/343; IC à 95 %, 87,9­94,0), et C. perfringens (61,5 %, 211/343; IC à 95 %, 56,3­66,7) était le plus répandu. Une co-infection avec deux à cinq pathogènes a été trouvée dans 67,3 % (231/343; IC à 95 %, 62,4­72,3) des échantillons étudiés. Il y avait des différences significatives (p < 0,05) dans les taux positifs de Cryptosporidium spp. et d'E. coli diarrhéogènes entre les fermes, les groupes d'âge et les saisons. Deux espèces de Cryptosporidium (C. parvum et C. bovis) et cinq sous-types de gp60 de C. parvum (IIdA15G1, IIdA20G1, IIdA19G1, IIdA14G1 et un nouveau, IIdA13G1) ont été identifiés. Deux assemblages (assemblage E et assemblage zoonotique A) de G. duodenalis et six génotypes ITS d'E. bieneusi (J, Henan-IV, EbpC, I, EbpA et ESH-01) ont été observés. Quatre gènes de virulence (eaeA, stx1, stx2 et st) d'E. coli diarrhéogènes et un type de toxine (type A) de C. perfringens ont été détectés. Notre étude enrichit les connaissances sur les caractères et le potentiel zoonotique des agents pathogènes liés à la diarrhée chez les veaux laitiers.

Virulence, multiple drug resistance, and biofilm-formation in Salmonella species isolated from layer, broiler, and dual-purpose indigenous chickens.

Globally, the significant risk to food safety and public health posed by antimicrobial-resistant foodborne Salmonella pathogens is driven by the utilization of in-feed antibiotics, with variations in usage across poultry production systems. The current study investigated the occurrence of virulence, antimicrobial resistant profiles, and biofilm-forming potentials of Salmonella isolates sourced from different chicken types. A total of 75 cloacal faecal samples were collected using sterile swabs from layer, broiler, and indigenous chickens across 15 poultry farms (five farms per chicken type). The samples were analysed for the presence of Salmonella spp. using species-specific PCR analysis. Out of the 150 presumptive isolates, a large proportion (82; 55%) were confirmed as Salmonella species, comprising the serovars S. typhimurium (49%) and S. enteritidis (30%) while 21% were uncategorised. Based on phenotypic antibiotic susceptibility test, the Salmonella isolates were most often resistant to erythromycin (62%), tetracycline (59%), and trimethoprim (32%). The dominant multiple antibiotic resistance phenotypes were SXT-W-TE (16%), E-W-TE (10%), AML-E-TE (10%), E-SXT-W-TE (13%), and AMP-AML-E-SXT-W-TE (10%). Genotypic assessment of antibiotic resistance genes revealed that isolates harboured the ant (52%), tet (A) (46%), sui1 (13%), sui2 (14%), and tet (B) (9%) determinants. Major virulence genes comprising the invasion gene spiC, the SPI-3 encoded protein (misL) that is associated with the establishment of chronic infections and host specificity as well as the SPI-4 encoded orfL that facilitates adhesion, autotransportation and colonisation were detected in 26%, 16%, and 14% of the isolates respectively. There was no significant difference on the proportion of Salmonella species and the occurrence of virulence and antimicrobial resistance determinants among Salmonella isolates obtained from different chicken types. In addition, neither the chicken type nor incubation temperature influenced the potential of the Salmonella isolates to form biofilms, although a large proportion (62%) exhibited weak to strong biofilm-forming potentials. Moderate to high proportions of antimicrobial resistant pathogenic Salmonella serovars were detected in the study but these did not vary with poultry production systems.

Examination of Salmonella Prevalence in Pigs Through Rye-Based Feeding and Coarser Feed Structure Under Field Conditions.

INTRODUCTION: Salmonellosis is the second most commonly occurring bacterial zoonosis in Germany. Rye in pig feeding offers new possibilities for addressing that issue due to its high content of non-starch polysaccharides (NSPs). These are fermented in the intestinal tract to specific fermentation products, which seem to have bacteriolytic effects against Salmonella. A coarse feed structure can display synergistic effects. METHODS: Seven conventional pig fattening farms increased the rye content (40%-70%) while limiting the amount of fine particles (maximum of 20% ≤0.25 mm). Samples from pigs were tested for Salmonella antibodies and compared with samples from 167 farms without any changes to the feed. RESULTS: Rye-based diets had a significant (p < 0.05) impact on Salmonella antibody (percentage optical density [OD%]) detection. In this study, it became apparent that significantly fewer positive OD% values could be detected due to the increase in rye compared to farms that did not change the feed (Farm 6 P0: 35.45 ± 36.18; P1: 15.48 ± 16.98; P2: 9.36 ± 8.17). An elimination of Salmonella could not be achieved, but especially farms with high antibody counts were able to strongly reduce those in both phases consecutively (Farm 5 P0: 35.17 ± 35.53; P1: 18.56a ± 20.96; P2: 13.38a ± 18.99). That was different on farms without adapted feeding, where an increase in Salmonella antibodies was observed (P0: 17.38 ± 22.21; P1: 20.12 ± 25.39; P2: 18.12 ± 25.44). CONCLUSION: By increasing the proportion of rye and limiting the proportion of fine particles in the feed, Salmonella antibodies (OD% values) in meat juice and blood can be significantly reduced, especially on farms with an initially high incidence of Salmonella. If that is implemented in feeding across the board on farms, an improvement in food safety and a decreased risk of zoonosis can be expected.

Salmonella associated with agricultural animals exhibit diverse evolutionary rates and show evidence of recent clonal expansion.

Most foodborne salmonellosis outbreaks are linked to agricultural animal products with a few serovars accounting for most Salmonella isolated from specific animal products, suggesting an adaptation to the corresponding animal hosts and their respective environments. Here, we utilized whole-genome sequence (WGS) data to analyze the evolution and population genetics of seven serovars frequently isolated from ground beef (Montevideo, Cerro, and Dublin), chicken (Kentucky, Infantis, and Enteritidis), and turkey (Reading) in the United States. In addition, publicly available metadata were used to characterize major clades within each serovar with regard to public health significance. Except for Dublin, all serovars were polyphyletic, comprising 2-6 phylogenetic groups. Further partitioning of the phylogenies identified 25 major clades, including 12 associated with animal or environmental niches. These 12 clades differed in evolutionary parameters (e.g., substitution rates) as well as public health relevant characteristics (e.g., association with human illness, antimicrobial resistance). Overall, our results highlight several critical trends: (i) the Salmonella generation time appears to be more dependent on source than serovar and (ii) all serovars contain clades and sub-clades that are estimated to have emerged after the year 1940 and that are enriched for isolates associated with humans, agricultural animals, antimicrobial resistance (AMR), and/or specific geographical regions. These findings suggest that serotyping alone does not provide enough resolution to differentiate isolates that may have evolved independently, present distinct geographic distribution and host association, and possibly have distinct public health significance. IMPORTANCE: Non-typhoidal Salmonella are major foodborne bacterial pathogens estimated to cause more than one million illnesses, thousands of hospitalizations, and hundreds of deaths annually in the United States. More than 70% of Salmonella outbreaks in the United States have been associated with agricultural animals. Certain serovars include persistent strains that have repeatedly contaminated beef, chicken, and turkey, causing outbreaks and sporadic cases over many years. These persistent strains represent a particular challenge to public health, as they are genetically clonal and widespread, making it difficult to differentiate distinct outbreak and contamination events using whole-genome sequence (WGS)-based subtyping methods (e.g., core genome allelic typing). Our results indicate that a phylogenetic approach is needed to investigate persistent strains and suggest that the association between a Salmonella serovar and an agricultural animal is driven by the expansion of clonal subtypes that likely became adapted to specific animals and associated environments.

Prevalence, antimicrobial resistance, and genomic characterization of Salmonella strains isolated in Hangzhou, China: a two-year study.

This study explored the molecular epidemiology and resistance mechanisms of 271 non-duplicate Salmonella enterica (S. enterica) strains, isolated mainly from adults (209/271) in a tertiary hospital in Hangzhou between 2020 and 2021. Through whole-genome sequencing and bioinformatics, the bacterial strains were classified into 46 serotypes and 54 sequence types (ST), with S. Enteritidis, S. 1,4,[5],12:i:-, and S. Typhimurium being the most prevalent serotypes and ST11, ST34, and ST19 the most common STs. The strains isolated from adults were primarily S. Enteritidis (59/209), while from children were mainly S. 1,4,[5],12:i:- (20/62). Worryingly, 12.55% strains were multi-drug resistant (MDR), with resistance rates to cefepime (FEP), ceftazidime (CAZ), ceftriaxone (CRO) and cefotaxime (CTX) of 7.38%, 9.23%, 15.87% and 16.24%, respectively, and resistance rates to levofloxacin (LEV) and ciprofloxacin (CIP) of 8.49% and 19.19%, respectively. It is worth noting that the resistance rates of CRO and CTX in children reached 30.65%. A total of 34 strains carried extended-spectrum ß-lactamase (ESBL) genes, dominated by blaCTX-M-65 (13/34) and blaCTX-M-55 (12/34); it is notable that one strain of S. Saintpaul carried both blaCTX-M-27 and blaCTX-M-55. The resistance mechanism to cephalosporins was mainly due to ESBL genes (20/43), and other genes included AmpC and ß-lactamase genes. The strains resistant to quinolones mainly carried qnrS1 (27/53), and others included qnrB6, aac(6')-Ib-cr, and mutations in gyrA and parC. One strain did not carry common quinolone resistance genes but had a parC (p.T57S) mutation to cause CIP resistance. This research provides vital insights into the molecular epidemiology and resistance mechanisms of clinical S. enterica, implicating possible infection control strategies.

A multi-provincial outbreak of Salmonella Newport infections associated with red onions: A report of the largest Salmonella outbreak in Canada in over 20 years.

An investigation into an outbreak of Salmonella Newport infections in Canada was initiated in July 2020. Cases were identified across several provinces through whole-genome sequencing (WGS). Exposure data were gathered through case interviews. Traceback investigations were conducted using receipts, invoices, import documentation, and menus. A total of 515 cases were identified in seven provinces, related by 0-6 whole-genome multi-locus sequence typing (wgMLST) allele differences. The median age of cases was 40 (range 1-100), 54% were female, 19% were hospitalized, and three deaths were reported. Forty-eight location-specific case sub-clusters were identified in restaurants, grocery stores, and congregate living facilities. Of the 414 cases with exposure information available, 71% (295) had reported eating onions the week prior to becoming ill, and 80% of those cases who reported eating onions, reported red onion specifically. The traceback investigation identified red onions from Grower A in California, USA, as the likely source of the outbreak, and the first of many food recall warnings was issued on 30 July 2020. Salmonella was not detected in any tested food or environmental samples. This paper summarizes the collaborative efforts undertaken to investigate and control the largest Salmonella outbreak in Canada in over 20 years.

Diarrhoeagenic Escherichia coli and Salmonella spp. Contamination of Food and Water Consumed by Children with Diarrhoea in Maputo, Mozambique.

In Mozambique, about 500,000 cases of diarrhoea were caused by foodborne pathogens in 2018. A review of the epidemiology of diarrhoea in children under five showed a high disease burden. This study aimed to identify Diarrhoeagenic Escherichia coli (DEC) and Salmonella spp. contamination of food and water in urban and rural areas of Maputo consumed by children under five with diarrhoea. One hundred and eighty-six children with diarrhoea were selected from Primeiro de Maio and Marracuene Health Care Centres from the Kamaxakeni and Marracuene districts, respectively. Food (n = 167) and water (n = 100) samples were collected in children's households for diarrhoeagenic bacterial identification. Interviews were conducted using a semi-structured questionnaire to collect data about demographics and foods consumed a week before the children's diarrhoea episodes. The prevalence of both DEC and Salmonella spp. was 9.8% in food and 5.4% in water samples. DEC was most prevalent in cereals (urban = 2.8%; rural = 2.4%) and water samples (urban = 1.4%; rural = 3.3%). Salmonella spp. was mainly detected in cereals (urban = 0.7%; rural = 0.8%). Diarrhoeagenic pathogens were associated with the type of food frequently consumed by children under five years with diarrhoea (infant formula, fruit puree, ready-to-eat meals, and bottled water), while the association with demographics was absent. We found that the infant foods consumed by children with diarrhoea are associated with DEC and Salmonella spp., and the prevalence of these contaminants is higher in the rural (8.9%) than in the urban area (6.3%), showing the need for caregiver education on food handling practices.

Outbreak-associated Salmonella Baildon found in wastewater demonstrates how sewage monitoring can supplement traditional disease surveillance.

Non-typhoidal Salmonella is a common cause of gastroenteritis worldwide, but current non-typhoidal Salmonella surveillance is suboptimal. Here, we evaluated the utility of wastewater monitoring to enhance traditional surveillance for this foodborne pathogen. In June 2022, we tested raw sewage collected twice a week from two treatment plants in central Pennsylvania for non-typhoidal Salmonella and characterized isolates using whole-genome sequencing. We recovered 43 Salmonella isolates from wastewater samples, differentiated by genomic analysis into seven serovars: 16 Panama (37.2%), 9 Senftenberg (20.9%), 8 Baildon (18.6%), and 3 or fewer of four other serovars. We assessed genetic relatedness and epidemiologic links between these wastewater isolates with those from patients with salmonellosis. All S. Baildon isolates from wastewater were genetically similar to those associated with a known contemporaneous salmonellosis outbreak. S. Baildon from wastewater and 42 outbreak-related isolates in the national outbreak detection database had the same core genome multilocus sequence typing, and outbreak code differed by zero or one single polynucleotide polymorphism. One of the 42 outbreak-related isolates was obtained from a patient residing in the wastewater sample collection catchment area, which serves approximately 17000 people. S. Baildon is a rare serovar (reported in <1% cases nationally, over five years). Our study underscores the value of monitoring sewage from a defined population to supplement traditional surveillance methods for the evidence of Salmonella infections and to determine the extent of outbreaks.IMPORTANCEDuring the COVID-19 pandemic, monitoring for SARS-CoV-2 in wastewater was highly effective in identifying the variants of concern earlier than clinical surveillance methods. Here, we show that monitoring domestic sewage can also augment traditional reporting of foodborne illnesses to public health authorities. Our study detected multiple Salmonella enterica serovars in samples from two wastewater treatment plants in central Pennsylvania. Using whole-genome sequencing, we demonstrated that the isolates of variant S. Baildon clustered with those from a foodborne salmonellosis outbreak that occurred in a similar time frame. Cases were primarily from Pennsylvania, and one individual lived within the wastewater treatment catchment area. This study highlights the effectiveness of domestic sewage testing as a proactive public health strategy to track and respond to infectious disease outbreaks.

[The application and reflection of emerging molecular detection technologies in Salmonella detection].

Salmonella is an important foodborne pathogen and one of the main causes of diarrhea. Every year, about 550 million people suffer from diarrhea due to Salmonella infection, of which about 230 000 die. It has become a major global public safety issue. The application fields of Salmonella detection involve food safety, water quality monitoring, animal husbandry, public health monitoring, and medical diagnosis. The detection requirements mainly come from three aspects: pathogen identification, serotype identification, drug resistance and virulence identification. In recent years, the detection technology for Salmonella has made rapid progress, especially the emergence and development of emerging molecular detection technologies, providing new perspectives for Salmonella detection in different scenarios. However, due to the diversity of Salmonella serotypes and the complexity of detection scenarios, existing detection technologies still have some pain points (such as long detection time, cumbersome operation steps, low scene adaptability, etc.). This article will elaborate on the application of several emerging molecular detection technologies with distinct characteristics, such as CRISPR Cas technology, digital PCR technology, sequencing technology, and microfluidic technology, in Salmonella detection. It aims to provide a reference for the development and improvement of Salmonella detection technology and the establishment of infection warning and control systems.

Surveillance of household foodborne disease outbreaks in Zhejiang Province, China, 2010-2022.

BACKGROUND: Foodborne diseases are a growing public health concern worldwide and households are a common setting. This study aimed to explore the epidemiological characteristics of household foodborne disease outbreaks in Zhejiang Province and propose targeted prevention and control measures. METHODS: Descriptive statistical methods were used to analyze household foodborne disease outbreak data collected from the Foodborne Disease Outbreaks Surveillance System in Zhejiang Province from 2010 to 2022. RESULTS: Household foodborne disease outbreaks showed an upward trend during the study period (Cox-Staurt trend test, p = 0.01563 < 0.05). These outbreaks mainly occurred from June to September, with 62.08% (352/567) of all reported outbreaks. The number of reported outbreaks varied in 11 prefectures, with a maximum of 100 and a minimum of only 7. Household foodborne disease outbreaks had a wide spectrum of etiologic factors. Mushroom toxins accounted for the largest proportion of all etiologies (43.39 %) and caused the highest proportion of hospitalization (54.18%) and death (78.26%). Such outbreaks are caused by accidently eating wild poisonous mushrooms. Bacterial infection (16.23%) was the second most common etiology, with Salmonella spp. and Vibrio parahaemolyticus being the primary pathogens. These outbreaks were caused by improper storage, improper processing or a combination of factors, and the foods involved were mainly aquatic animals, eggs and cooked meat. Other identified etiologies included plant toxins (9.52%), chemicals (7.23%), animal toxins (3.70%), and viruses (1.76%). Among the above-mentioned etiologies, mushroom toxins, bacteria, and animal toxins had seasonal characteristics. Analysis of regions and etiologies revealed that the proportion of various etiologies was different in 11 prefectures. Wild mushrooms (43.39%), aquatic animals (9.88%), and toxic plants (8.47%) were the top three foods involved in these outbreaks. The most common factors contributing to household foodborne disease outbreaks were inedibility and misuse (59.08%), followed by multiple factors (7.58%), improper storage (7.41%), and improper processing (7.41%). CONCLUSIONS: Household foodborne disease outbreaks were closely related to the lack of knowledge regarding foodborne disease prevention. Therefore, public health agencies should strengthen residents' surveillance and health education to improve food safety awareness and effectively reduce foodborne diseases in households. In addition, timely publicity and early warning by relevant government departments, the introduction of standards to control the contamination of pathogenic bacteria in raw materials, and strengthened supervision of the sale of substances that may cause health hazards, such as poisonous mushrooms and nitrites, will also help reduce such outbreaks.

Prevalence of Salmonella spp. in meat, seafood, and leafy green vegetables from local markets and vegetable farms in Phnom Penh, Cambodia.

Salmonella is a major bacterial concern for public health globally. Although there are limited documentation on the prevalence of Salmonella species in Cambodia's food chain, some reports indicate that salmonellosis is a severe gastrointestinal infection in its population and especially in children. To investigate the presence of Salmonella spp., 285 food samples (75 meat, 50 seafood, and 160 leafy green vegetable samples) were randomly collected from various local markets in Phnom Penh capital and nearby farms in Cambodia. Concurrently, field observations were conducted to collect data on food hygiene and practices among the relevant actors. All food samples were analyzed using bacterial culture and plate counts, and the findings were confirmed serially with biochemical, serological, and PCR tests. The observational data on food hygiene and practices from farm to market revealed that the spread of Salmonella in the food-value chain from farm to market could pose health risks to consumers. The overall prevalence of Salmonella spp. was 48.4% (138/285), while the prevalence in meat, seafood, and vegetables was 71% (53/75), 64% (32/50), and 33% (53/160), respectively. Mean Salmonella plate count ranged from 1.2 to 7.40 log10 CFU/g, and there was no significant difference in bacterial counts between meat, seafood, and vegetable samples (p > 0.05). The most common serogroups among the isolated Salmonella spp. were B and C. These results suggest that a large proportion of meat, seafood, and vegetable products sold at local markets in Phnom Penh are contaminated with Salmonella spp. This is likely linked to inadequate hygiene and sanitation practices, including handling, storage, and preservation conditions. Observations on farms suggested that the prevalence of Salmonella in vegetables sold at the market could be linked to contamination relating to agricultural practices. Thus, controlling the spread of foodborne salmonellosis through the food-value chain from farms and retailers to consumers is warranted to enhance food safety in Cambodia.

Prevalence and characteristics of ESBL-producing Salmonella in Weifang, China.

This study examined the prevalence and antibiotic resistance pattern of blaCTX-M extended-spectrum ß-lactamase positive Salmonella species isolated from a hospital in Weifang. Salmonella strains were isolated from hospitalized patients from January 2018 to April 2023. Whole-genome sequencing was performed by Illumina platform. CTX-M-producing Salmonella were identified by Comprehensive Antibiotic Research Database (CARD). Strain susceptibility to six antimicrobial agents was assessed by BD Phoenix™ M50 System. MLST analysis confirmed sequence types and additionally, serotypes were determined by SeqSero2. Genetic environments of blaCTX-M genes were analyzed by Isfinder and BLASTn. Single nucleotide polymorphisms were used to construct a phylogenetic tree to analyze homology. A total of 34 CTX-M-producing Salmonella were detected. The most prevalent serotype was Salmonella enterica subsp. enterica 1,4,[5],12:i:- (14/34, 41.18%), belonging to ST34, followed by Salmonella Enteritidis (10/34, 29.41%), belonging to ST11. The highest resistance rate was detected to ampicillin (97.06%), followed by ceftriaxone (94.12%) and ceftazidime (58.83%). In CTX-M-producing Salmonella five types of blaCTX-M genes were identified, the most prevalent was blaCTX-M-55 (47.06%, 16/34), followed by blaCTX-M-14, blaCTX-M-65, blaCTX-M-125, and blaCTX-M-27 at 26.47% (9/34), 11.77% (4/34), 8.82% (3/34), and 5.88% (2/34), respectively. Apart from blaCTX-M, 40 antibiotic resistance genes were also detected, conveying resistance to multiple drugs and the most frequent genes were namely, mcr-1.1, aph(6)-Id, aph(3″)-Ib, oqxAB, qnrB6, qnrS1. According to genetic environment analysis, the insertion sequence ISEcp1 was prevalent upstream of the blaCTX-M gene. Our study demonstrates that multiple resistance genes are carried by clinical isolates of Salmonella spp. however, the dominant ESBL genotype is CTX-M-55, that is associated with ISEcp1.

Exploring antibiotic-induced persister formation and bacterial persistence genes in clinical isolates from Burkina Faso.

BACKGROUND: In addition to antibiotic resistance, persistence is another cause of treatment failure in bacterial infections, representing a significant public health concern. Due to a lack of adequate data on clinical isolates, this study was initiated to investigate persistence in clinical isolates in Burkina Faso. METHODS: Eighty (80) clinical isolates, including 32 Pseudomonas aeruginosa, 41 Staphylococcus aureus, and 7 Salmonella sp. obtained from clinical laboratories in Burkina Faso, were analyzed to assess their susceptibility to ciprofloxacin and gentamicin, as well as to determine the presence of persistence genes. The effects of ciprofloxacin and gentamicin on persister formation were evaluated by conducting colony counts at 1, 3, 5, 7, and 20 h after exposing the bacteria to high concentrations of these antibiotics. RESULTS: Results showed high sensitivity to both antibiotics (72.5% for ciprofloxacin and 82.5% for gentamicin). Persister formation occurred in Staphylococcus aureus with gentamicin and in Salmonella sp. with ciprofloxacin, while Pseudomonas aeruginosa did not form persisters. The mazF gene was found in 28.13% of P. aeruginosa and 2.44% of S. aureus isolates, and the hipA gene in 28.57% of Salmonella sp. None of the relE1 or relE2 genes were detected. CONCLUSIONS: The study revealed high sensitivity in clinical bacterial isolates to ciprofloxacin and gentamicin. Staphylococcus aureus and Salmonella sp. showed persister formation under antibiotic stress, with low frequencies of the studied persistence genes. These findings enhance understanding of clinical bacterial behavior and inform strategies against antibiotic-resistant infections.

Biotemplated Janus Magnetic Microrobots Based on Diatomite for Highly Efficient Detection of Salmonella.

Foodborne illnesses caused by Salmonella bacteria pose a significant threat to public health. It is still challenging to detect them effectively. Herein, biotemplated Janus disk-shaped magnetic microrobots (BJDMs) based on diatomite are developed for the highly efficient detection of Salmonella in milk. The BJDMs were loaded with aptamer, which can be magnetically actuated in the swarm to capture Salmonella in a linear range of 5.8 × 102 to 5.8 × 105 CFU/mL in 30 min, with a detection limit as low as 58 CFU/mL. In addition, the silica surface of BJDMs exhibited a large specific surface area to adsorb DNA from captured Salmonella, and the specificity was also confirmed via tests of a mixture of diverse foodborne bacteria. These diatomite-based microrobots hold the advantages of mass production and low cost and could also be extended toward the detection of other types of bacterial toxins via loading different probes. Therefore, this work offers a reliable strategy to construct robust platforms for rapid biological detection in practical applications of food safety.