Endoprotein-activating DNAzyme assay for nucleic acid extraction- and amplification-free detection of viable pathogenic bacteria.

Pathogenic bacteria in food or environment, can pose threats to public health, highlighting the requirement of tools for rapid and accurate detection of viable pathogenic bacteria. Herein, we report a sequential endoprotein RNase H2-activating DNAzyme assay (termed epDNAzyme) that enables nucleic acid extraction- and amplification-free detection of viable Salmonella enterica (S. enterica). The direct detection allows for a rapid detection of viable S. enterica within 25 min. Besides, the assay, based on sequential reporting strategy, circumvents internal modifications in the DNAzyme's active domain and improve its catalytic activity. The multiple-turnover DNAzyme cutting and the enhanced catalytic activity of DNAzyme render the epDNAzyme assay to be highly sensitive, and enables the detection of 190 CFU/mL and 0.1% viable S. enterica. The assay has been utilized to detect S. enterica contamination in food and clinical samples, indicating its potential as a promising tool for monitoring pathogen-associated biosafety.

Ceftriaxone-resistant Salmonella enterica serovar Newport: a case report from South India.

Salmonella enterica serovar Newport is a human pathogen underreported in most developing countries. It is known for causing gastroenteritis and extraintestinal infections. In this case report, we report the case of ceftriaxone-resistant Salmonella enterica serovar Newport from South India, causing acute gastroenteritis in a sixty-year-old female patient having a history of antimicrobial therapy and recent hospital admission. Serovar Newport, especially among antibiotic-exposed patients, poses a significant public health threat due to its ability to acquire multidrug resistance. This emphasizes the necessity for robust surveillance and monitoring of nontyphoidal Salmonella infections, particularly given the limited data on serovar Newport in India. Vigilance in clinical practice and public health initiatives is crucial to effectively address the emergence and spread of multidrug-resistant strains.

Colistin-, cefepime-, and levofloxacin-resistant Salmonella enterica serovars isolated from Egyptian chicken carcasses.

OBJECTIVES: The emergence of multidrug-resistant (MDR) Salmonella strains, especially resistant ones toward critically important antimicrobial classes such as fluoroquinolones and third- and fourth-generation cephalosporins, is a growing public health concern. The current study, therefore, aimed to determine the prevalence, and existence of virulence genes (invA, stn, and spvC genes), antimicrobial resistance profiles, and the presence of ß-lactamase resistance genes (blaOXA, blaCTX-M1, blaSHV, and blaTEM) in Salmonella strains isolated from native chicken carcasses in Egypt marketed in Mansoura, Egypt, as well as spotlight the risk of isolated MDR, colistin-, cefepime-, and levofloxacin-resistant Salmonella enterica serovars to public health. METHODS: One hundred fifty freshly dressed native chicken carcasses were collected from different poultry shops in Mansoura City, Egypt between July 2022 and November 2022. Salmonella isolation was performed using standard bacteriological techniques, including pre-enrichment in buffered peptone water (BPW), selective enrichment in Rappaport Vassiliadis broth (RVS), and cultivating on the surface of xylose-lysine-desoxycholate (XLD) agar. All suspected Salmonella colonies were subjected to biochemical tests, serological identification using slide agglutination test, and Polymerase Chain Reaction (PCR) targeting the invasion A gene (invA; Salmonella marker gene). Afterward, all molecularly verified isolates were screened for the presence of virulence genes (stn and spvC). The antimicrobial susceptibility testing for isolated Salmonella strains towards the 16 antimicrobial agents tested was analyzed by Kirby-Bauer disc diffusion method, except for colistin, in which the minimum inhibition concentration (MIC) was determined by broth microdilution technique. Furthermore, 82 cefotaxime-resistant Salmonella isolates were tested using multiplex PCR targeting the ß-lactamase resistance genes, including blaOXA, blaCTX-M1, blaSHV, and blaTEM genes. RESULTS: Salmonella enterica species were molecularly confirmed via the invA Salmonella marker gene in 18% (27/150) of the freshly dressed native chicken carcasses. Twelve Salmonella serotypes were identified among 129 confirmed Salmonella isolates with the most predominant serotypes were S. Kentucky, S. Enteritidis, S. Typhimurium, and S. Molade with an incidence of 19.4% (25/129), 17.1% (22/129), 17.1% (22/129), and 10.9% (14/129), respectively. All the identified Salmonella isolates (n = 129) were positive for both invA and stn genes, while only 31.8% (41/129) of isolates were positive for the spvC gene. One hundred twenty-one (93.8%) of the 129 Salmonella-verified isolates were resistant to at least three antibiotics. Interestingly, 3.9%, 14.7%, and 75.2% of isolates were categorized into pan-drug-resistant, extensively drug-resistant, and multidrug-resistant, respectively. The average MAR index for the 129 isolates tested was 0.505. Exactly, 82.2%, 82.2%, 63.6%, 51.9%, 50.4%, 48.8%, 11.6%, and 10.1% of isolated Salmonella strains were resistant to cefepime, colistin, cefotaxime, ceftazidime/clavulanic acid, levofloxacin, ciprofloxacin, azithromycin, and meropenem, respectively. Thirty-one out (37.8%) of the 82 cefotaxime-resistant Salmonella isolates were ß-lactamase producers with the blaTEM as the most predominant ß-lactamase resistance gene, followed by blaCTX-M1 and blaOXA genes, which were detected in 21, 16, and 14 isolates respectively). CONCLUSION: The high prevalence of MDR-, colistin-, cefepime-, and levofloxacin-resistant Salmonella serovars among Salmonella isolates from native chicken is alarming as these antimicrobials are critically important in treating severe salmonellosis cases and boost the urgent need for controlling antibiotic usage in veterinary and human medicine to protect public health.

High prevalence and genomic features of multidrug-resistant Salmonella enterica isolated from chilled broiler chicken on retail sale in the United Arab Emirates.

Non-typhoidal Salmonella represents a significant global concern for food safety and One Health. Despite the United Arab Emirates (UAE) being a leading consumer of chicken meat globally, there is a lack of comprehensive understanding regarding the prevalence and genomic characteristics of Salmonella within the country. This study aims to address this gap by conducting a thorough analysis of Salmonella prevalence, antimicrobial resistance, and genomic profiles of isolates obtained from whole broiler carcasses retailed under chilled conditions in the UAE. Our findings reveal that Salmonella was detected in 41.2 % (130/315) of the sampled chilled broiler carcasses, with notable variability observed among samples sourced from six different companies. Phenotypic antimicrobial resistance (AMR) testing, among 105 isolates, highlighted high resistance rates to tetracycline (97.1 %), nalidixic acid (93.3 %), ampicillin (92.4 %), azithromycin (75.2 %), ciprofloxacin (63.8 %), and ceftriaxone (54.3 %). Furthermore, a concerning 99 % (104/105) of the isolates exhibited multidrug resistance. Whole-genome sequencing (WGS) of 60 isolates identified five serovars, with S. infantis/Sequence Type (ST) 32 (55 %) and S. Minnesota/ST-458 (28.3 %) being the most prevalent. WGS analysis unveiled 34 genes associated with antimicrobial resistance, including mcr-1.1 (only in two isolates), conferring resistance to colistin. The two major serovars, Infantis and Minnesota, exhibited significant variation (P-values <0.001) in the distribution of major AMR genes (aadA1, blaCMY-2, blaSHV-12, qnrB19, qnrS1, sul1, and sul2). Notably, the gene qacEdelta, conferring resistance to quaternary ammonium compounds commonly found in disinfectants, was universally present in all S. Infantis isolates (n = 33), compared to only one S. Minnesota isolate. Additionally, all S. Infantis isolates harbored the IncFIB (pN55391) plasmid replicon type. Major serovars exhibited distinct distributions of antimicrobial resistance genes, underscoring the importance of serovar-specific surveillance. These findings emphasize the critical need for continuous surveillance and intervention measures to address Salmonella contamination risks in poultry products, providing valuable insights for public health and regulatory strategies not only in the UAE but also globally.

Benchmarking short and long read polishing tools for nanopore assemblies: achieving near-perfect genomes for outbreak isolates.

BACKGROUND: Oxford Nanopore provides high throughput sequencing platforms able to reconstruct complete bacterial genomes with 99.95% accuracy. However, even small levels of error can obscure the phylogenetic relationships between closely related isolates. Polishing tools have been developed to correct these errors, but it is uncertain if they obtain the accuracy needed for the high-resolution source tracking of foodborne illness outbreaks. RESULTS: We tested 132 combinations of assembly and short- and long-read polishing tools to assess their accuracy for reconstructing the genome sequences of 15 highly similar Salmonella enterica serovar Newport isolates from a 2020 onion outbreak. While long-read polishing alone improved accuracy, near perfect accuracy (99.9999% accuracy or ~ 5 nucleotide errors across the 4.8 Mbp genome, excluding low confidence regions) was only obtained by pipelines that combined both long- and short-read polishing tools. Notably, medaka was a more accurate and efficient long-read polisher than Racon. Among short-read polishers, NextPolish showed the highest accuracy, but Pilon, Polypolish, and POLCA performed similarly. Among the 5 best performing pipelines, polishing with medaka followed by NextPolish was the most common combination. Importantly, the order of polishing tools mattered i.e., using less accurate tools after more accurate ones introduced errors. Indels in homopolymers and repetitive regions, where the short reads could not be uniquely mapped, remained the most challenging errors to correct. CONCLUSIONS: Short reads are still needed to correct errors in nanopore sequenced assemblies to obtain the accuracy required for source tracking investigations. Our granular assessment of the performance of the polishing pipelines allowed us to suggest best practices for tool users and areas for improvement for tool developers.

Virulence factors of Salmonella spp. isolated from free-living grass snakes Natrix natrix.

Salmonellosis associated with reptiles is a well-researched topic, particularly in China and the United States, but it occurs less frequently in Europe. The growth of the human population and changes in the environment could potentially increase the interaction between humans and free-living reptiles, which are an unidentified source of Salmonella species. In this study, we sought to explore this issue by comparing the microbiota of free-living European grass snakes, scientifically known as Natrix natrix, with that of captive banded water snakes, or Nerodia fasciata. We were able to isolate 27 strains of Salmonella species from cloacal swabs of 59 N. natrix and 3 strains from 10 N. fasciata. Our findings revealed that free-living snakes can carry strains of Salmonella species that are resistant to normal human serum (NHS). In contrast, all the Salmonella species strains isolated from N. fasciata were sensitive to the action of the NHS, further supporting our findings. We identified two serovars from N. natrix: Salmonella enterica subspecies diarizonae and S. enterica subspecies houtenae. Additionally, we identified three different virulotypes (VT) with invA, sipB, prgH, orgA, tolC, iroN, sitC, sifA, sopB, spiA, cdtB and msgA genes, and ß-galactosidase synthesised by 23 serovars. The identification of Salmonella species in terms of their VT is a relatively unknown aspect of their pathology. This can be specific to the serovar and pathovar and could be a result of adaptation to a new host or environment.

A one-pot loop-mediated isothermal amplification platform using fluorescent gold nanoclusters for rapid and naked-eye pathogen detection.

Loop-mediated isothermal amplification (LAMP) is a rapid and sensitive nucleic acid testing method for pathogen detection, yet the absence of a straightforward readout strategy remains challenging. We've successfully designed polyethyleneimine-stabilized gold nanoclusters (PEI-AuNCs) as a cationic AuNCs indicator tailored for distinguishing LAMP results, enabling direct visual inspection under UV light. Positive LAMP reactions with PEI-AuNCs, in combination with magnesium pyrophosphate crystals, yield red-fluorescent bulk precipitates visible to the naked eye. To address contamination concerns, we introduced a one-pot reaction by incorporating AuNCs into the lid recess. This one-pot LAMP assay demonstrates exceptional detection capability, identifying Salmonella enterica at concentrations as low as 101 CFU/mL within approximately 50 min, excluding nucleic acid extraction. The platform's versatility, achieved through customizable primers, positions it as a promising molecular diagnostic tool for rapid and visual pathogen detection across scientific disciplines.

Emergence of Salmonella Infantis carrying the pESI-like plasmid from eggs in egg grading and packing plants in Korea.

The plasmid of emerging S. Infantis (pESI) or pESI-like plasmid in Salmonella enterica Infantis are consistently reported in poultry and humans worldwide. However, there has been limited research on these plasmids of S. Infantis isolated from eggs. Therefore, this study aimed to analyze the prevalence and characteristics of S. Infantis carrying the pESI-like plasmid from eggs in egg grading and packing plants. In this study, the pESI-like plasmid was only detected in 18 (78.3%) of 23 S. Infantis isolates, and it was absent in the other 9 Salmonella serovars. In particular, S. Infantis isolates carrying the pESI-like plasmid showed the significantly higher resistance to ß-lactams, phenicols, cephams, aminoglycosides, quinolones, sulfonamides, and tetracyclines than Salmonella isolates without the pESI-like plasmid (p < 0.05). Moreover, all S. Infantis isolates carrying the pESI-like plasmid were identified as extended-spectrum ß-lactamase (ESBL) producer, harboring the blaCTX-M-65 and blaTEM-1 genes, and carried non-ß-lactamase resistance genes (ant(3'')-Ia, aph(4)-Ia, aac(3)-IVa, aph(3')-Ic, sul1, tetA, dfrA14, and floR) against five antimicrobial classes. However, all isolates without the pESI-like plasmid only carried the blaTEM-1 gene among the ß-lactamase genes, and either had no non-ß-lactamase resistance genes or harbored non-ß-lactamase resistance genes against one or two antimicrobial classes. Furthermore, all S. Infantis isolates carrying the pESI-like plasmid carried class 1 and 2 integrons and the aadA1 gene cassette, but none of the other isolates without the pESI-like plasmid harbored integrons. In particular, D87Y substitution in the gyrA gene and IncP replicon type were observed in all the S. Infantis isolates carrying the pESI-like plasmid but not in the S. Infantis isolates without the pESI-like plasmid. The distribution of pulsotypes between pESI-positive and pESI-negative S. Infantis isolates was clearly distinguished, but all S. Infantis isolates were classified as sequence type 32, regardless of whether they carried the pESI-like plasmid. This study is the first to report the characteristics of S. Infantis carrying the pESI-like plasmid isolated from eggs and can provide valuable information for formulating strategies to control the spread of Salmonella in the egg industry worldwide.

Does Salmonella diarizonae 58:r:z53 Isolated from a Mallard Duck Pose a Threat to Human Health?

Salmonella diarizonae (IIIb) is frequently isolated from reptiles and less frequently from birds and mammals. However, its isolation from invasive human infections has not been widely reported. Migratory mallard ducks are excellent bioindicators of pathogen presence and pathogen antibiotic resistance (AMR). We present the first isolation from a mallard duck in central Europe of the antibiotic-resistant Salmonella enterica subsp. diarizonae with the unique antigenic pattern 58:r:z53 and report its whole-genome sequencing, serosequencing, and genotyping, which enabled the prediction of its pathogenicity and comparison with phenotypic AMR. The isolated strain was highly similar to S. diarizonae isolated from humans and food. Twenty-four AMR genes were detected, including those encoding aminoglycoside, fluoroquinolone, macrolide, carbapenem, tetracycline, cephalosporin, nitroimidazole, peptide antibiotic, and disinfecting agent/antiseptic resistance. Six Salmonella pathogenicity islands were found (SPI-1, SPI-2, SPI-3, SPI-5, SPI-9, and SPI-13). An iron transport system was detected in SPI-1 centisome C63PI. Plasmid profile analyses showed three to be present. Sequence mutations in the invA and invF genes were noted, which truncated and elongated the proteins, respectively. The strain also harbored genes encoding type-III secretion-system effector proteins and many virulence factors found in S. diarizonae associated with human infections. This study aims to elucidate the AMR and virulence genes in S. enterica subsp. diarizonae that may most seriously threaten human health.

A biphasic accelerated strand exchange amplification strategy for culture-independent and rapid detection of Salmonella enterica in food samples.

Salmonella enterica is a common foodborne pathogen that can cause food poisoning in humans. The organism also infects and causes disease in animals. Rapid and sensitive detection of S. enterica is essential to prevent the spread of this pathogen. Traditional technologies for the extraction and detection of this pathogen from complex food matrices are cumbersome and time-consuming. In this study, we introduced a novel strategy of biphasic assay integrated with an accelerated strand exchange amplification (ASEA) method for efficient detection of S. enterica without culture or other extraction procedures. Food samples are rapidly dried, resulting in a physical fluidic network inside the dried food matrix, which allows polymerases and primers to access the target DNA and initiate ASEA. The dried food matrix is defined as the solid phase, while amplification products are enriched in the supernatant (liquid phase) and generate fluorescence signals. The analytical performances demonstrated that this strategy was able to specifically identify S. enterica and did not show any cross-reaction with other common foodborne pathogens. For artificially spiked food samples, the strategy can detect 5.0 × 101 CFU mL-1S. enterica in milk, 1.0 × 102 CFU g-1 in duck, scallop or lettuce, and 1.0 × 103 CFU g-1 in either oyster or cucumber samples without pre-enrichment of the target pathogen. We further validated the strategy using 82 real food samples, and this strategy showed 92% sensitivity. The entire detection process can be finished, sample-to-answer, within 50 min, dramatically decreasing the detection time. Therefore, we believe that the proposed method enables rapid and sensitive detection of S. enterica and holds great promise for the food safety industry.

Genomic investigation of Salmonella enterica Serovar Welikade from a pediatric diarrhea case first time in Shanghai, China.

BACKGROUND: Salmonella, an important foodborne pathogen, was estimated to be responsible for 95.1 million cases and 50,771 deaths worldwide. Sixteen serovars were responsible for approximately 80% of Salmonella infections in humans in China, and infections caused by a few uncommon serovars have been reported in recent years, though not with S. Welikade. This study reports the first clinical case caused by S. Welikade in China and places Chinese S. Welikade isolates in the context of global isolates via genomic analysis. For comparison, S. Welikade isolates were also screened in the Chinese Local Surveillance System for Salmonella (CLSSS). The minimum inhibitory concentrations (MICs) of 28 antimicrobial agents were determined using the broth microdilution method. The isolates were sequenced on an Illumina platform to identify antimicrobial resistance genes, virulence genes, and phylogenetic relationships. RESULTS: The S. Welikade isolate (Sal097) was isolated from a two-year-old boy with acute gastroenteritis in 2021. Along with the other two isolates found in CLSSS, the three Chinese isolates were susceptible to all the examined antimicrobial agents, and their sequence types (STs) were ST5123 (n = 2) and ST3774 (n = 1). Single nucleotide polymorphism (SNP)-based phylogenetic analysis revealed that global S. Welikade strains can be divided into four groups, and these three Chinese isolates were assigned to B (n = 2; Sal097 and XXB1016) and C (n = 1; XXB700). In Group B, the two Chinese ST5123 isolates were closely clustered with three UK ST5123 isolates. In Group C, the Chinese isolate was closely related to the other 12 ST3774 isolates. The number of virulence genes in the S. Welikade isolates ranged from 59 to 152. The galF gene was only present in Group A, the pipB2 gene was only absent from Group A, the avrA gene was only absent from Group B, and the allB, sseK1, sspH2, STM0287, and tlde1 were found only within Group C and D isolates. There were 15 loci unique to the Sal097 isolate. CONCLUSION: This study is the first to characterize and investigate clinical S. Welikade isolates in China. Responsible for a pediatric case of gastroenteritis in 2021, the clinical isolate harbored no antimicrobial resistance and belonged to phylogenetic Group B of global S. Welikade genomes.

Development of a decision support tool to compare diagnostic strategies for establishing the herd status for infectious diseases: An example with Salmonella Dublin infection in dairies.

The diagnosis of infectious diseases at herd level can be challenging as different stakeholders can have conflicting priorities. The current study proposes a "proof of concept" of an approach that considers a reasonable number of criteria to rank plausible diagnostic strategies using multi-criteria decision analysis (MCDA) methods. The example of Salmonella Dublin diagnostic in Québec dairy herds is presented according to two epidemiological contexts: (i) in herds with no history of S. Dublin infection and absence of clinical signs, (ii) in herds with a previous history of infection, but absence of clinical signs at the moment of testing. Multiple multiparty exchanges were conducted to determine: 1) stakeholders' groups; 2) the decision problem; 3) solutions to the problem (options) or diagnostic strategies to be ordered; 4) criteria and indicators; 5) criteria weights; 6) the construction of a performance matrix for each option; 7) the multi-criteria analyses using the visual preference ranking organization method for enrichment of evaluations approach; 8) the sensitivity analyses, and 9) the final decision. A total of nine people from four Québec's organizations (the dairy producers provincial association along with the DHI company, the ministry of agriculture, the association of veterinary practitioners, and experts in epidemiology) composed the MCDA team. The decision problem was "What is the optimal diagnostic strategy for establishing the status of a dairy herd for S. Dublin infection when there are no clinical signs of infection?". Fourteen diagnostic strategies composed of the three following parameters were considered: 1) biological samples (bulk tank milk or blood from 10 heifers aged over three months); 2) sampling frequencies (one to three samples collection visits); 3) case definitions to conclude to a positive status using imperfect milk- or blood-ELISA tests. The top-ranking diagnostic strategy was the same in the two contexts: testing the bulk tank milk and the blood samples, all samples collected during one visit and the herd being assigned a S. Dublin positive status if one sample is ELISA-positive. The final decision favored the top-ranking option for both contexts. This MCDA approach and its application to S. Dublin infection in dairy herds allowed a consensual, rational, and transparent ranking of feasible diagnostic strategies while taking into account the diagnostic tests accuracy, socio-economic, logistic, and perception considerations of the key actors in the dairy industry. This promising tool can be applied to other infectious diseases that lack a well-established diagnostic procedure to define a herd status.

Occurrence, antimicrobial resistance, and molecular characterization of Salmonella enterica from chicken products and human in Wasit Governorate of Iraq.

Background: Salmonella infections are considered the most common foodborne pathogens responsible for zoonotic infections and food poisoning in humans and animal species such as birds. Antimicrobial resistance is considered a global anxiety because it causes human public health repercussions, as well as leads to an increase in animal morbidity and death. Aim: The aims of this study are the isolation and identification of Salmonella enterica, as well as to investigate the antimicrobial susceptibility test (AST) and the molecular characteristics using polymerase chain reaction (PCR) and sequences for isolates from chicken products (eggs, livers, and minced meat) and human in the Wasit Governorate of Iraq. Methods: A total of 300 samples (150 chicken product samples including eggs, livers, and minced meat, and 150 human fecal samples) were collected from the Wasit governorate of Iraq from January to December 2022. The bacterial isolation was done according to recommendations of ISO 6579 standard and the Global Foodborne Infections Network laboratory protocol. Serotyping test and AST were done by using 19 antibiotic agents according to the recommendations of the Clinical and Laboratory Standards Institute, 2022 by using disc diffusion susceptibility test and Vitik 2 test. Finally, the suspected isolates were confirmed using the conventional PCR method and sequencing for a unique rRNA gene. Results: The results showed that the isolation percentage of S. enterica in chicken products was 8.66% (12% eggs, 6% livers, and 8% minced meat), while in humans it was 4.6%. Also, showed 100% of Salmonella typhi in humans. While, in chicken eggs S. typhi, Salmonella typhimurium, and Salmonella enteritidis were 50%, 33.33%, and 16.66%, respectively. Also, showed 100% of S. typhimurium in both livers and minced meat. The AST in human isolates showed resistance to Ampicillin, Cefotaxime, Ceftazidime, Cefepime, Amikacin, Gentamicin, Ciprofloxacin, Norfloxacin, and Ceftriaxone, while no resistance to Amoxicillin, Pipracillin, Ertapenem, Imipenem, Meropenem, Fosfomycin, Nitrofurantoin, Trimethoprim, Azithromycin, and Tetracycline. In chicken products, isolates were resistant with different percentages to Amikacin, Gentamicin, Tetracycline, Ciprofloxacin, Norfloxacin, Nitrofurantoin, Ampicillin, Cefotaxime, Ceftazidime, Cefepime, and Trimethoprim; while no resistance to Amoxicillin, Pipracillin, Ertapenem, Imipenem, Meropenem, Fosfomycin, Azithromycin, and Ceftriaxone. Sequencing by using rRNA gene was done for four PCR products. Conclusion: This study showed the presence of genetic mutations for S. enterica which led to variations in the molecular characteristics, and antimicrobial drug resistance of S. enterica isolated from chicken products and humans.

Curing of a field strain of Salmonella enterica serovar Infantis isolated from poultry from its highly stable pESI like plasmid.

Salmonella enterica serovar Infantis (S. infantis) is an important emerging pathogen, associated with poultry and poultry products and related to an increasing number of human infections in many countries. A concerning trend among S. infantis isolates is the presence of plasmid-mediated multidrug resistance. In many instances, the genes responsible for this resistance are carried on a megaplasmid known as the plasmid of emerging S. infantis (pESI) or pESI like plasmids. Plasmids can be remarkably stable due to the presence of multiple replicons and post-segregational killing systems (PSKs), which contribute to their maintenance within bacterial populations. To enhance our understanding of S. infantis and its multidrug resistance determinants toward the development of new vaccination strategies, we have devised a new method for targeted plasmid curing. This approach effectively overcomes plasmid addiction by leveraging the temporal overproduction of specific antitoxins coupled with the deletion of the partition region. By employing this strategy, we successfully generated a plasmid-free strain from a field isolate derived from S. infantis 119,944. This method provides valuable tools for studying S. infantis and its plasmid-borne multidrug resistance mechanisms and can be easily adopted for plasmid curing from other related bacteria.

Immobilization of a broad host range phage on the peroxidase-like Fe-MOF for colorimetric determination of multiple Salmonella enterica strains in food.

A broad host range phage-based nanozyme (Fe-MOF@SalmpYZU47) was prepared for colorimetric detection of multiple Salmonella enterica strains. The isolation of a broad host range phage (SalmpYZU47) capable of infecting multiple S. enterica strains was achieved. Then, it was directly immobilized onto the Fe-MOF to prepare Fe-MOF@SalmpYZU47, exhibiting peroxidase-like activity. The peroxidase-like activity can be specifically inhibited by multiple S. enterica strains, benefiting from the broad host range capture ability of Fe-MOF@SalmpYZU47. Based on it, a colorimetric detection approach was developed for S. enterica in the range from 1.0 × 102 to 1.0 × 108 CFU mL-1, achieving a low limit of detection (LOD) of 11 CFU mL-1. The Fe-MOF@SalmpYZU47 was utilized for detecting S. enterica in authentic food samples, achieving recoveries ranging from 91.88 to 105.34%. Hence, our proposed broad host range phage-based nanozyme exhibits significant potential for application in the colorimetric detection of pathogenic bacteria.

Multi-locus sequence typing, antimicrobials resistance and virulence profiles of Salmonella enterica isolated from bovine carcasses in Minas Gerais state, Brazil.

The aim of this study was to identify virulence and antimicrobial resistance profiles and determine the sequence type (ST) by multilocus sequence typing (MLST) of Salmonella enterica isolates from bovine carcasses from slaughterhouse located in Minas Gerais state, Brazil, and its relationship with bovine isolates obtained on the American continent based on sequence type profile. The MLST results were compared with all Salmonella STs associated with cattle on American continent, and a multi-locus sequence tree (MS tree) was built. Among the 17 S. enterica isolates, five ST profiles identified, and ST10 were the most frequent, grouping seven (41.2%) isolates. The isolates presented 11 different profiles of virulence genes, and six different antibiotics resistance profiles. The survey on Enterobase platform showed 333 Salmonella STs from American continent, grouped into four different clusters. Most of the isolates in the present study (13/17), were concentrated in a single cluster (L4) composed by 74 STs. As a conclusion, five different STs were identified, with ST10 being the most common. The isolates showed great diversity of virulence genes and antibiotics resistance profiles. Most of the isolates of this study were grouped into a single cluster composed by 74 STs formed by bovine isolates obtained on the American continent.

Trends in antimicrobial susceptibility pattern of Salmonella species isolated from bacteremia patients at a tertiary care center in Northern India.

The study was done to assess the antimicrobial susceptibility pattern among Salmonella enterica serovars causing bacteremia in Northern India. In this observational study, blood samples positive for Salmonella enterica serovars from January 2021 to April 2023 were studied. Species identification was done using MALDI-ToF MS. Serotyping was done using slide agglutination method. Antimicrobial susceptibility was interpreted as per the CLSI guidelines. During the study period, 32 Salmonella enterica serovars were isolated. Salmonella enterica serovar Typhi was the predominant serovar, followed by Salmonella enterica serovar Paratyphi A. All isolates were susceptible to ceftriaxone, chloramphenicol, co-trimoxazole and cefotaxime. Pefloxacin showed 100% resistance. Resistance to nalidixic acid was found in 81.2% isolates. Of the isolates resistant to nalidixic acid, 19(73.08%) isolates were resistant to ciprofloxacin also. This changing susceptibility pattern necessitates continuous surveillance of antibiogram of Salmonella isolates to rationalize the treatment protocols for invasive salmonellosis and prevent emergence of resistant strains.

Colonization, spread and persistence of Salmonella (Typhimurium, Infantis and Reading) in internal organs of broilers.

Transfer of Salmonella to internal organs of broilers over a 35 d grow-out period was evaluated. A total of 360 one-day old chicks were placed in 18 floor pens of 3 groups with 6 replicate pens each. On d 0, broilers were orally challenged with a cocktail of Salmonella (equal population of marked serovars; nalidixic acid-resistant S. Typhimurium, rifampicin-resistant S. Infantis, and kanamycin-resistant S. Reading) to have 3 groups: L (low; ∼2 log CFU/bird); M (medium; ∼5 log CFU/bird); and H (High; ∼8 log CFU/bird). On d 2, 7 and 35, 4 birds/pen were euthanized and ceca, liver, and spleen samples were collected aseptically. Gizzard samples (4/pen) were collected on d 35. The concentration of Salmonella in liver and spleen were transformed to binary outcomes (positive and negative) and fitted in glm function of R using cecal Salmonella concentrations (log CFU/g) and inoculation doses (L, M, and H) as inputs. On d 2, H group showed greater (P ≤ 0.05) cecal colonization of all 3 serovars compared to L and M groups. However, M group showed greater (P ≤ 0.05) colonization of all 3 serovars in the liver and spleen compared to L group. Salmonella colonization increased linearly in the ceca and quadratically in the liver and spleen with increasing challenge dose (P ≤ 0.05). On d 35, L group had greater (P ≤ 0.05) S. Infantis colonization in the ceca and liver compared to M and H groups (P ≤ 0.05). Moreover, within each group on d 35, the concentration of S. Reading was greater than those of S. Typhimurium and S. Infantis for all 3 doses in the ceca and high dose in the liver and gizzard (P ≤ 0.05). Salmonella colonization diminished in the ceca, liver, and spleen during grow-out from d 0 to d 35 (P ≤ 0.05). On d 35, birds challenged with different doses of Salmonella cocktail showed a similar total Salmonella spp. population in the ceca (ca. 3.14 log CFU/g), liver (ca. 0.54 log CFU/g), spleen (ca. 0.31 log CFU/g), and gizzard (ca. 0.42 log CFU/g). Estimates from the fitted logistic model showed that one log CFU/g increase in cecal Salmonella concentration will result in an increase in relative risk of liver and spleen being Salmonella-positive by 4.02 and 3.40 times (P ≤ 0.01), respectively. Broilers from H or M group had a lower risk (28 and 23%) of being Salmonella-positive in the liver compared to the L group when the cecal Salmonella concentration is the same (P ≤ 0.05). Oral challenge of broilers with Salmonella spp. with various doses resulted in linear or quadratic increases in Salmonella colonization in the internal organs during early age and these populations decreased during grow-out (d 35). This research can provide guidance on practices to effectively mitigate the risk of Salmonella from chicken parts and enhance public health.

Comparative genomics reveals high genetic similarity among strains of Salmonella enterica serovar Infantis isolated from multiple sources in Brazil.

Background: Salmonella enterica serovar Infantis (Salmonella Infantis) is a zoonotic, ubiquitous and foodborne pathogen of worldwide distribution. Despite Brazil's relevance as a major meat exporter, few studies were conducted to characterize strains of this serovar by genomic analyses in this country. Therefore, this study aimed to assess the diversity of 80 Salmonella Infantis strains isolated from veterinary, food and human sources in Brazil between 2013 and 2018 by comparative genomic analyses. Additional genomes of non-Brazilian countries (n = 18) were included for comparison purposes in some analyses. Methods: Analyses of whole-genome multi-locus sequence typing (wgMLST), using PGAdb-builder, and of fragmented genomes, using Gegenees, were conducted to compare the 80 Brazilian strains to the 18 non-Brazilian genomes. Pangenome analyses and calculations were performed for all Salmonella Infantis genomes analyzed. The presence of prophages was determined using PHASTER for the 80 Brazilian strains. The genome plasticity using BLAST Ring Image Generator (BRIG) and gene synteny using Mauve were evaluated for 20 selected Salmonella Infantis genomes from Brazil and ten from non-Brazilian countries. Unique orthologous protein clusters were searched in ten selected Salmonella Infantis genomes from Brazil and ten from non-Brazilian countries. Results: wgMLST and Gegenees showed a high genomic similarity among some Brazilian Salmonella Infantis genomes, and also the correlation of some clusters with non-Brazilian genomes. Gegenees also showed an overall similarity >91% among all Salmonella Infantis genomes. Pangenome calculations revealed an open pangenome for all Salmonella Infantis subsets analyzed and a high gene content in the core genomes. Fifteen types of prophages were detected among 97.5% of the Brazilian strains. BRIG and Mauve demonstrated a high structural similarity among the Brazilian and non-Brazilian isolates. Unique orthologous protein clusters related to biological processes, molecular functions, and cellular components were detected among Brazilian and non-Brazilian genomes. Conclusion: The results presented using different genomic approaches emphasized the significant genomic similarity among Brazilian Salmonella Infantis genomes analyzed, suggesting wide distribution of closely related genotypes among diverse sources in Brazil. The data generated contributed to novel information regarding the genomic diversity of Brazilian and non-Brazilian Salmonella Infantis in comparison. The different genetically related subtypes of Salmonella Infantis from Brazil can either occur exclusively within the country, or also in other countries, suggesting that some exportation of the Brazilian genotypes may have already occurred.

Centralized industrialization of pork in Europe and America contributes to the global spread of Salmonella enterica.

Salmonella enterica causes severe food-borne infections through contamination of the food supply chain. Its evolution has been associated with human activities, especially animal husbandry. Advances in intensive farming and global transportation have substantially reshaped the pig industry, but their impact on the evolution of associated zoonotic pathogens such as S. enterica remains unresolved. Here we investigated the population fluctuation, accumulation of antimicrobial resistance genes and international serovar Choleraesuis transmission of nine pig-enriched S. enterica populations comprising more than 9,000 genomes. Most changes were found to be attributable to the developments of the modern pig industry. All pig-enriched salmonellae experienced host transfers in pigs and/or population expansions over the past century, with pigs and pork having become the main sources of S. enterica transmissions to other hosts. Overall, our analysis revealed strong associations between the transmission of pig-enriched salmonellae and the global pork trade.