The transcriptional regulation of the horizontally acquired iron uptake system, yersiniabactin and its contribution to oxidative stress tolerance and pathogenicity of globally emerging salmonella strains.

The bacterial species Salmonella enterica (S. enterica) is a highly diverse pathogen containing more than 2600 distinct serovars, which can infect a wide range of animal and human hosts. Recent global emergence of multidrug resistant strains, from serovars Infantis and Muenchen is associated with acquisition of the epidemic megaplasmid, pESI that augments antimicrobial resistance and pathogenicity. One of the main pESI's virulence factors is the potent iron uptake system, yersiniabactin encoded by fyuA, irp2-irp1-ybtUTE, ybtA, and ybtPQXS gene cluster. Here we show that yersiniabactin, has an underappreciated distribution among different S. enterica serovars and subspecies, integrated in their chromosome or carried by different conjugative plasmids, including pESI. While the genetic organization and the coding sequence of the yersiniabactin genes are generally conserved, a 201-bp insertion sequence upstream to ybtA, was identified in pESI. Despite this insertion, pESI-encoded yersiniabactin is regulated by YbtA and the ancestral Ferric Uptake Regulator (Fur), which binds directly to the ybtA and irp2 promoters. Furthermore, we show that yersiniabactin genes are specifically induced during the mid-late logarithmic growth phase and in response to iron-starvation or hydrogen peroxide. Concurring, yersiniabactin was found to play a previously unknown role in oxidative stress tolerance and to enhance intestinal colonization of S. Infantis in mice. These results indicate that yersiniabactin contributes to Salmonella fitness and pathogenicity in vivo and is likely to play a role in the rapid dissemination of pESI among globally emerging Salmonella lineages.

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.

Oral vaccination of young broilers with a live Salmonella Typhimurium vaccine reduces caecal and internal organ colonization following a Salmonella Infantis challenge in a seeder-bird model.

Poultry products are an important source of foodborne Salmonella infections in humans. Amongst these, the prevalence of S. Infantis is rising. In this study, the protection efficacy of an authorized live-attenuated S. Typhimurium vaccine against S. Infantis, was examined using a seeder-bird model in broilers. Vaccinated birds displayed a significantly lower colonization of S. Infantis bacteria in the caeca compared to the non-vaccinated counterparts (P = 0.017), with no significant differences observed in the spleen among the groups, three days post-infection. Thirty-two days post-infection, the disparity in average S. Infantis concentration between all-vaccinated and non-vaccinated birds was significant in both caeca (P = 0.0003) and spleen (P = 0.0002). Interestingly, a third group, consisting of seeder birds that were not vaccinated but housed with vaccinated penmates, exhibited significantly lower S. Infantis levels in both caeca (P = 0.0014) and spleen (P < 0.0001) compared to the non-vaccinated group. These findings underscore the potential of a live-attenuated S. Typhimurium vaccine administered to 2-day-old chicks in conferring protection against S. Infantis in broilers up to slaughter age.

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.

CRISPR and CRISPR-MVLST reveal conserved spacer distribution and high similarity among Salmonella enterica serovar Infantis genomes from Brazil and other countries.

Salmonella enterica serovar Infantis (S. Infantis) is a globally distributed non-typhoid serovar infecting humans and food-producing animals. Considering the zoonotic potential and public health importance of this serovar, strategies to characterizing, monitor and control this pathogen are of great importance. This study aimed to determine the genetic relatedness of 80 Brazilian S. Infantis genomes in comparison to 40 non-Brazilian genomes from 14 countries using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-Multi-Locus Virulence Sequence Typing (CRISPR-MVLST). CRISPR spacers were searched using CRISPR-Cas++ and fimH and sseL alleles using BLAST and MEGA X. Results were analyzed using BioNumerics 7.6 in order to obtain similarity dendrograms. A total of 23 CRISPR1 and 11 CRISPR2 alleles formed by 37 and 26 types of spacers, respectively, were detected. MVLST revealed the presence of five fimH and three sseL alleles. CRISPR's similarity dendrogram showed 32 strain subtypes, with an overall similarity ≥ 78.6. The CRISPR-MVLST similarity dendrogram showed 37 subtypes, with an overall similarity ≥ 79.2. In conclusion, S. Infantis strains isolated from diverse sources in Brazil and other countries presented a high genetic similarity according to CRISPR and CRISPR-MVLST, regardless of their source, year, and/or place of isolation. These results suggest that both methods might be useful for molecular typing S. Infantis strains using WGS data.

Genomic characterisation of the population structure and antibiotic resistance of Salmonella enterica serovar Infantis in Chile, 2009-2022.

Background: Multidrug-resistant (MDR) Salmonella Infantis has disseminated worldwide, mainly linked to the consumption of poultry products. Evidence shows dissemination of this pathogen in Chile; however, studies are primarily limited to phenotypic data or involve few isolates. As human cases of Salmonella Infantis infections have substantially increased in recent years, this study aimed to characterise the genomic epidemiology and antimicrobial-resistance profiles of isolates obtained from different sources, aiming to inform effective surveillance and control measures. Methods: We sequenced 396 Salmonella Infantis genomes and analysed them with all publicly available genomes of this pathogen from Chile (440 genomes in total), representing isolates from environmental, food, animal, and human sources obtained from 2009 to 2022. Based on bioinformatic and phenotypic methods, we assessed the population structure, dissemination among different niches, and antimicrobial resistance (AMR) profiles of Salmonella Infantis in the country. Findings: The genomic and phylogenetic analyses showed that Salmonella Infantis from Chile comprised several clusters of highly related isolates dominated by sequence type 32. The HC20_343 cluster grouped an important proportion of all isolates. This was the only cluster associated with pESI-like megaplasmids, and up to 12 acquired AMR genes/mutations predicted to result in an MDR phenotype. Accordingly, antimicrobial-susceptibility testing revealed a strong concordance between the AMR genetic determinants and their matching phenotypic expression, indicating that a significant proportion of HC20_343 isolates produce extended-spectrum ß-lactamases and have intermediate fluoroquinolone resistance. HC20_343 Salmonella Infantis were spread among environmental, animal, food, and human niches, showing a close relationship between isolates from different years and sources, and a low intra-source genomic diversity. Interpretation: Our findings show a widespread dissemination of MDR Salmonella Infantis from the HC20_343 cluster in Chile. The high proportion of isolates with resistance to first-line antibiotics and the evidence of active transmission between the environment, animals, food, and humans highlight the urgency of improved surveillance and control measures in the country. As HC20_343 isolates predominate in the Americas, our results suggest a high prevalence of ESBL-producing Salmonella Infantis with intermediate fluoroquinolone resistance in the continent. Funding: Partially supported by the Food and Drug Administration (FDA) of the U.S. Department of Health and Human Services as part of an award, FDU001818, with 30% percent funded by FDA/HHS; and by Agencia de Investigación y Desarrollo de Chile (ANID) through FONDECYT de Postdoctorado Folio 3230796 and Folio 3210317, FONDECYT Regular Folio 1231082, and ANID-Millennium Science Initiative Program-ICN2021_044.

Uncommon Salmonella Infantis Variants with Incomplete Antigenic Formula in the Poultry Food Chain, Italy.

Uncommon Salmonella Infantis variants displaying only flagellar antigens phenotypically showed identical incomplete antigenic formula but differed by molecular serotyping. Although most formed rough colonies, all shared antimicrobial resistances and the presence of usg gene with wild-type Salmonella Infantis. Moreover, they were undistinguishable wild-type Salmonella Infantis by whole-genome sequencing.

Emergence of Salmonella Infantis carrying the pESI megaplasmid in commercial farms of five major integrated broiler operations in Korea.

Considering Salmonella transmission occurs through several routes in integrated broiler operations, control of nontyphoidal Salmonella in commercial farms is essential. This study aimed to compare the distribution of persistent Salmonella serovars in environments and dead chickens between 5 major integrated broiler operations in Korea. The prevalence of Salmonella-positive farms in dust prior to placement by operations was 0 to 25%, but the prevalence in dust and feces at the time of depletion was increased to 16.7 to 41.7% and 16.7 to 66.7%, respectively. Moreover, the prevalence of farms with Salmonella in chickens that died within 1 week old and at 4 to 5 weeks old ranged from 8.3 to 58.3% and 16.7 to 41.7%, respectively. The prevalence of Salmonella enterica serovar Infantis-positive farms in dust prior to placement and in chickens that died within 1 week old was 5.2 and 3.4%, respectively, but the prevalence in dust and feces at the time of depletion and in chickens that died at 4 to 5 weeks old was significantly increased to 27.6, 41.4, and 20.7%, respectively (P < 0.05). Interestingly, the plasmid of emerging S. Infantis (pESI) was only identified in S. Infantis, and the prevalence of multidrug-resistance was significantly higher in pESI-positive S. Infantis (99.2%) than in pESI-negative S. Infantis (6.7%) (P < 0.05). The distribution of pulsotypes between pESI-positive and pESI-negative S. Infantis were varied, but a majority of S. Infantis were clustered only 2 pulsotypes. Moreover, pESI-positive S. Infantis harbored more virulence factors than pESI-negative S. Infantis. This study is the first report on characteristics of S. Infantis carrying the pESI plasmid in commercial broiler farms in Korea.

From farm to fork: Spread of a multidrug resistant Salmonella Infantis clone encoding blaCTX-M-1 on pESI-like plasmids in Central Italy.

Salmonella enterica subsp. enterica serovar Infantis (S. Infantis) is one of the "top five Salmonella serovars" of clinical significance in the European Union (EU). Antimicrobial resistant and extended spectrum ß-lactamase (ESBL) AmpC-producing S. Infantis have been described in food production systems and human clinical samples in Italy. Recently, an increase of MDR S. Infantis carrying blaCTX-M genes involved in 3rd generation cephalosporin resistance was noticed in the EU, including Italy, mainly due to the spread of S. Infantis harboring a pESI-like plasmid. The aim of this study was to investigate the occurrence of the S. Infantis pESI-like plasmid among antibiotic resistant S. Infantis strains isolated at different points of the food chain, and to provide a phylogenetic analysis to gain further insight on their transmission pathways from 'farm to fork'. MDR S. Infantis strains (n. 35) isolated from 2016 to 2021 at different stages of the food chain (animals, food, food-related environments, and humans) were investigated with in depth molecular characterization using real-time PCR, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and whole genome sequencing (WGS). Our study reported the occurrence of S. Infantis strains harboring pESI-like plasmids, carrying blaCTX-M-1 genes, in Central Italy, at different sampling points along the food chain. Results confirmed the presence of a plasmid with a molecular size around 224-310 kb, thus consistent with the pESI-like, in 97 % of the 35 samples investigated. Two variants of S. Infantis pESI-like IncFIB(K)_1_Kpn3 were detected, one associated with the European clone carrying blaCTX-M-1 (21 isolates) and the other associated with U.S. isolates carrying blaCTX-M-65 (2 isolates, pESI-like U.S. variant). The majority was resistant to 3rd generation cephalosporins but none of the strains tested positive for the carbapenemase encoding genes. A total of 118 virulence genes were identified in isolates harboring the pESI-like plasmid. cgMLST and SNP-based analysis revealed the presence of one main cluster, composed by strains isolated from the environment, animals, food and humans. The results of this investigation underline the importance of phylogenetic studies to monitor and understand pathogen and AMR spread in a One Health approach.

Occurrence of a New Variant of Salmonella Infantis Lacking Somatic Antigen.

Salmonella Infantis is one of the most frequent serovars reported in broilers and is also regularly identified in human salmonellosis cases, representing a relevant public health problem. In the laboratories of the Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata (IZSPB), six Salmonella Infantis strains with antigenic formula -:r:1,5 have been isolated from the litter and carcass of broilers between 2018 and 2022. The strains were investigated to evaluate their phenotype, antibiotic resistance and genomic profiles. Genomic analysis confirmed that the isolates belonged to the Infantis serotype and to the sequence type ST32. Moreover, all strains showed a multidrug-resistant (MDR) profile and were characterised by the presence of the IncFIB plasmid incompatibility group. Three strains had the blaCTX-M-1 gene, and one of them carried IncX1. The presence of this new variant of S. Infantis is particularly relevant because it could expand the landscape of the S. Infantis population. The absence of the somatic antigen could pose a problem in both isolation and serotyping and a consequent public health concern due to the spread of Salmonella infection.

SIMBA Method-Simultaneous Detection of Antimicrobial and Anti-biofilm Activity of New Compounds Using Salmonella Infantis.

The development of antimicrobial resistance and the formation of Salmonella biofilms are serious public health problems. For this reason, new natural compounds with antimicrobial and anti-biofilm activity are being sought, and wild fungi represent an untapped potential. Various extraction agents, including organic solvents and aqueous buffers, can be used to obtain bioactive compounds from natural sources. To evaluate their bioactivity, extensive screening studies are required to determine antimicrobial and anti-biofilm activity using methods such as broth microdilution or crystal violet assay, respectively, but none of these methods allow simultaneous evaluation of both activities against bacteria. Cold water extraction from wild fungi offers the advantage of extracting water-soluble compounds. The SIMultaneous detection of antiMicrobial and anti-Biofilm Activity (SIMBA) method combines the testing of both types of activity against bacteria with the evaluation of the 20 h growth curve of the Salmonella Infantis ZM9 strain determined with absorbance measurements at 600 nm in a 96-well plate. SIMBA method thus shortens the time to determine the bioactivity of extracts, reduces material consumption, and eliminates the need for additional reagents. SIMBA enables rapid selection of bioactive extracts for their fractionation and shortens the time to determine new natural products with antimicrobial and anti-biofilm activity. Graphical overview.

Holistic Strategies to Control Salmonella Infantis: An Emerging Challenge in the European Broiler Sector.

Salmonella spp. has been globally recognized as one of the leading causes of acute human bacterial gastroenteritis resulting from the consumption of animal-derived products. Salmonella Enteritidis, S. Typhimurium, and its monophasic variant are the main serovars responsible for human disease. However, a serovar known as S. Infantis has emerged as the fourth most prevalent serovar associated with human disease. A total of 95% of isolated S. Infantis serovars originate from broilers and their derived products. This serovar is strongly associated with an elevated antimicrobial (AMR) and multidrug resistance, a resistance to disinfectants, an increased tolerance to environmental mercury, a heightened virulence, and an enhanced ability to form biofilms and attach to host cells. Furthermore, this serovar harbors genes that confer resistance to colistin, a last-resort antibiotic in human medicine, and it has the potential to acquire additional transferable AMR against other critically important antimicrobials, posing a new and significant challenge to global public health. This review provides an overview of the current status of the S. Infantis serovar in the poultry sector, focusing on its key virulence factors, including its virulence genes, antimicrobial resistance, and biofilm formation. Additionally, novel holistic strategies for controlling S. Infantis along the entire food chain are presented in this review.

Polymerase chain reaction for the in vitro detection of the pESI plasmid associated with the globally circulating Salmonella Infantis outbreak strain.

A globally circulating strain of Salmonella enterica serotype Infantis containing the pESI plasmid has increased in prevalence in poultry meat samples and cases of human infections. In this study, a polymerase chain reaction (PCR) protocol was designed to detect the pESI plasmid and confirm the Infantis serotype of Salmonella isolates. Primers were tested bioinformatically to predict specificity, sensitivity, and precision. A total of 54 isolates of Salmonella serotypes Infantis, Senftenberg, and Alachua were tested, with and without the pESI plasmid carriage. Isolates of 31 additional serotypes were also screened to confirm specificity to Infantis. Specificity, sensitivity, and precision of each primer were >0.95. All isolates tested produced the expected band sizes. This PCR protocol provides a rapid and clear result for the detection of the pESI plasmid and serotype Infantis and will allow for the in vitro detection for epidemiological studies where whole-genome sequencing is not available.

A review of the global emergence of multidrug-resistant Salmonella enterica subsp. enterica Serovar Infantis.

Salmonella enterica serovar Infantis is an emergent foodborne and zoonotic Salmonella serovar with critical implications for global health. In recent years, the prevalence of S. Infantis infections has increased in the United States, Europe, and Latin America, due to contaminated chicken and other foods. An essential trait of S. Infantis is its resistance to multiple antibiotics, including the critically important third-generation cephalosporins and quinolones, undermining effective medical treatment, particularly in low-resource settings. We describe the emergence of multidrug-resistant (MDR) S. Infantis, focusing on humans, animals, the environment, and food. We conducted a systematic review (1979-2021), selected 183 studies, and analyzed the origin, source, antimicrobial resistance, and presence of a conjugative plasmid of emerging S. Infantis (pESI) in reported isolates. S. Infantis has been detected worldwide, with a substantial increase since 2011. We found the highest number of isolations in the Americas (42.9 %), Europe (29.8 %), Western Pacific (17.2 %), Eastern Mediterranean (6.6 %), Africa (3.4 %), and South-East Asia (0.1 %). S. Infantis showed MDR patterns and numerous resistant genes in all sources. The primary source of MDR S. Infantis is broiler and their meat; however, this emerging pathogen is also present in other reservoirs such as food, wildlife, and the environment. Clinical cases of MDR S. Infantis have been reported in children and adults. The global emergence of S. Infantis is related to a plasmid (pESI) with antibiotic and arsenic- and mercury-resistance genes. Additionally, a new megaplasmid (pESI-like), carrying blaCTX-M-65 and antibiotic-resistant genes reported in an ancestral version, was detected in the broiler, human, and chicken meat isolates. Strains harboring pESI-like were primarily observed in the Americas and Europe. MDR S. Infantis has spread globally, potentially becoming a major public health threat, particularly in low- and middle-income countries.

Whole-genome sequencing and phylogenetic analysis capture the emergence of a multi-drug resistant Salmonella enterica serovar Infantis clone from diagnostic animal samples in the United States.

Introduction: Salmonella enterica is a major cause of foodborne illness in the United States. A multi-drug resistant (MDR) emergent Salmonella Infantis (ESI) with a megaplasmid (pESI) was first identified in Israel and Italy and subsequently reported worldwide. The ESI clone carrying an extended spectrum ß-lactamase blaCTX-M-65 on a pESI-like plasmid and a mutation in the gyrA gene has recently been found in the United States in poultry meat. Methods: We analyzed the phenotypic and genotypic antimicrobial resistance, genomics and phylogeny of 200 S. infantis isolates from animal diagnostic samples. Results: Of these, 33.5% were resistant to at least one antimicrobial and 19.5% were multi-drug resistant (MDR). Eleven isolates from different animal sources were phenotypically and genetically similar to the ESI clone. These isolates had a D87Y mutation in the gyrA gene conferring reduced susceptibility to ciprofloxacin and harbored a combination of 6-10 resistance genes: blaCTX-M-65, aac(3)-IVa, aadA1, aph(4)-Ia, aph(3')-Ia, floR, sul1, dfrA14, tetA, and fosA. These 11 isolates carried class I and class II integrons and three virulence genes: sinH, involved in adhesion and invasion, ybtQ and ybtP, associated with iron transport. These isolates were also closely related to each other (separated by 7 to 27 SNPs) and phylogenetically related to the ESI clone recently found in the U.S. Discussion: This dataset captured the emergence of the MDR ESI clone in multiple animal species and the first report of a pESI-like plasmid in isolates from horses in the U.S.

Evaluation of ß-Lactamase Enzyme Activity in Outer Membrane Vesicles (OMVs) Isolated from Extended Spectrum ß-Lactamase (ESBL) Salmonella Infantis Strains.

Outer membrane vesicles (OMVs) are nanoparticles released by Gram-negative bacteria, which contain different cargo molecules and mediate several biological processes. Recent studies have shown that OMVs are involved in antibiotic-resistance (AR) mechanisms by including ß-lactamase enzymes in their lumen. Since no studies have as yet been conducted on Salmonella enterica subs. enterica serovar Infantis' OMVs, the aim of the work was to collect OMVs from five S. Infantis ß-lactam resistant strains isolated from a broiler meat production chain and to investigate whether ß-lactamase enzymes are included in OMVs during their biogenesis. OMVs were isolated by means of ultrafiltration and a Nitrocefin assay quantified the presence of ß-lactamase enzymes in the OMVs. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to identify the OMVs. The results showed that all strains release spherical OMVs, ranging from 60 to 230 nm. The Nitrocefin assay highlighted the presence of ß-lactamase enzymes within the OMVs. This suggests that ß-lactamase enzymes also get packaged into OMVs from bacterial periplasm during OMV biogenesis. An investigation into the possible role played by OMVs in AR mechanisms would open the door for an opportunity to develop new, therapeutic strategies.

Activity of plant essential oils against clinically and environmentally isolated Salmonella enterica serotypes: in vitro assays and molecular docking.

This research aimed to evaluate the antimicrobial activity of essential oils (EOs) against clinically and environmentally isolated Salmonella serotypes. Oregano, thyme, and grapefruit EO compounds were identified, and the antimicrobial activity was evaluated against the S. Saintpaul, Oranienburg, and Infantis serotypes. In addition, molecular docking was performed to explore the possible mechanisms between compounds of EOs with microbial enzymes. Thymol was the main compound identified in oregano (44.0%) and thyme (31%) EOs, while d-limonene was present in a greater proportion in grapefruit EO. Oregano EO had the highest antimicrobial activity, followed by thyme and grapefruit EOs. Oregano and thyme EOs illustrated a greater inhibitory capacity to all serotypes, particularly with the environmental S. Saintpaul. Oregano EO presented values of minimum inhibitory concentration (MIC) and minimum bactericidal concentration of 0.1 µL/mL for all serotypes, while thyme and grapefruit EOs presented MIC values of 0.1 µL/mL for the clinical serotypes S. Infantis and S. Oranienburg, respectively. Molecular docking analysis showed the optimal binding free energies for thymol and carvacrol with glucokinase, ATP-dependent-6-fructokinase, outer membrane porin C, and topoisomerase IV. Our results indicate that these EOs can inhibit clinically and environmentally isolated Salmonella serotypes and can be used as alternatives for developing natural food preservatives.

Buffered Peptone Water Formulation Does Not Influence Growth of pESI-positive Salmonella enterica Serovar Infantis.

Salmonella enterica is a major cause of human foodborne illness and is often attributed to poultry food sources. S. enterica serovar Infantis, specifically those carrying the pESI plasmid, has become a frequently isolated serotype from poultry meat samples at processing and has caused numerous recent human infections. In 2016, the USDA-Food Safety and Inspection Service changed the official sampling method for raw poultry products from BPW to using neutralizing BPW (nBPW) as the rinsing agent in order to prevent residual antimicrobial effects from acidifying and oxidizing processing aids. This change was contemporaneous to the emergence of pESI-positive ser. Infantis as a prevalent serovar in poultry, prompting some to question if nBPW could be selecting for this prevalent serovar. We performed two experiments: a comparison of ser. Infantis growth in BPW versus nBPW, and a simulation of regulatory sampling methods. We found that when inoculated into both broths, ser. Infantis initially grows slightly slower in nBPW than in BPW but little difference was seen in abundance after 6 h of growth. Additionally, the use of nBPW to simulate poultry rinse sample and overnight cold shipping to a regulatory lab did not affect the survival or subsequent growth of ser. Infantis in BPW. We concluded that the change in USDA-FSIS methodology to include nBPW in sampling procedures has likely not affected the emergence of S. ser. Infantis as a prevalent serovar in chicken and turkey meat product samples.

Antimicrobial and Antibiofilm Effect of Commonly Used Disinfectants on Salmonella Infantis Isolates.

Salmonella enterica subsp. enterica serovar Infantis is the most prevalent serovar in broilers and broiler meat in the European Union. The aim of our study was to test the biofilm formation and antimicrobial effect of disinfectants on genetically characterized S. Infantis isolates from poultry, food, and humans. For the biofilm formation under various temperature conditions (8 °C, 20 °C, and 28 °C) and incubation times (72 h and 168 h), the crystal violet staining method was used. The evaluation of the in vitro antimicrobial effect of Ecocid® S, ethanol, and hydrogen peroxide was determined using the broth microdilution method. The antibiofilm effect of subinhibitory concentration (1/8 MIC) of disinfectants was then tested on S. Infantis 323/19 strain that had the highest biofilm formation potential. Our results showed that the biofilm formation was strain-specific; however, it was higher at 20 °C and prolonged incubation time. Moreover, strains carrying a pESI plasmid showed higher biofilm formation potential. The antibiofilm potential of disinfectants on S. Infantis 323/19 strain at 20 °C was effective after a shorter incubation time. As shown in our study, more effective precautionary measures should be implemented to ensure biofilm prevention and removal in order to control the S. Infantis occurrence.

Evidence of structural rearrangements in ESBL-positive pESI(like) megaplasmids of S.Infantis.

The increasing prevalence of pESI(like)-positive, multidrug-resistant (MDR) S. Infantis in Europe is a cause of major concern. As previously demonstrated, the pESI(like) megaplasmid is not only a carrier of antimicrobial resistant (AMR) genes (at least tet, dfr, and sul genes), but also harbours several virulence and fitness genes, and toxin/antitoxin systems that enhance its persistence in the S. Infantis host. In this study, five prototype pESI(like) plasmids, of either CTX-M-1 or CTX-M-65 ESBL-producing strains, were long-read sequenced using Oxford Nanopore Technology (ONT), and their complete sequences were resolved. Comparison of the structure and gene content of the five sequenced plasmids, and further comparison with previously published pESI(like) sequences, indicated that although the sequence of such pESI(like) 'mosaic' plasmids remains almost identical, their structures appear different and composed of regions inserted or transposed after different events. The results obtained in this study are essential to better understand the plasticity and the evolution of the pESI(like) megaplasmid, and therefore to better address risk management options and policy decisions to fight against AMR and MDR in Salmonella and other food-borne pathogens. Graphical representation of the pESI-like plasmid complete sequence (ID 12037823/11). Block colours indicate the function of the genes: red: repB gene; pink: class I integrons (IntI); yellow; mobile elements; blue: resistance genes; green: toxin/anti-toxin systems; grey: mer operon; light green: genes involve in conjugation.