Investigation of prevalence and antimicrobial resistance of Salmonella in pet dogs and cats in Turkey.

BACKGROUND: Although salmonellosis is considered to be a foodborne zoonotic disease, pets can play a significant role in the dissemination of antimicrobial-resistant Salmonella organisms to humans because of close contact with their owners. OBJECTIVES: To determine the prevalence, risk factors, virulence factors, serotypes, and antimicrobial resistance profile of Salmonella in pet dogs and cats in Turkey and to assess the public health risk. Furthermore, to perform macroscopic comparison of lactic acid bacteria (LAB) in Salmonella-positive and Salmonella-negative animals. METHODS: International Standards Organization (ISO) 6579-1:2017 and Food and Drug Administration (FDA) methods were used to compare the effectiveness of culture methods in the identification of Salmonella in 348 rectal swabs. Positive isolates were serotyped using the slide agglutination method according to the White-Kauffmann-Le Minor scheme and the presence of virulence genes (invA and stn) were evaluated by polymerase chain reaction (PCR). Antimicrobial activity was tested by Kirby-Bauer disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI) guidelines. RESULTS: Salmonella prevalence was 5.73% (9/157) in dogs and 0.0% (0/191) in cats. Eight (8/9) isolates were cultured with the ISO method and 5 (5/9) isolates were cultured with the FDA method. Macroscopic results revealed that Salmonella agents had no effect on LAB. Three different serotypes were detected and all isolates were positive for virulence genes. Antibiotic resistance profiling indicated that 11.1% of the isolates were MDR and the highest resistance was found for ciprofloxacin. MDR-resistant S. Virchow and carbapenem-resistant S. Enteritidis were detected from dog isolates. There was a significant difference between raw meat consumption and Salmonella carriage (p < 0.01). CONCLUSIONS: Dogs could be potential carriers of Salmonella infection. The isolation of Salmonella in healthy dogs instead of dogs suffering from diarrhoea indicates that attention should be paid to asymptomatic carriage. The emergence of resistance among zoonotic Salmonella isolates poses a significant threat to public health.

Optimization of the molecular detection assay of Salmonella (MDS®) for drag swab samples of aviaries.

Detection of Salmonella sp. is important for the broiler chicken production chain because it is one microorganisms involved in food-borne diseases. Thus, this study performed the optimization of a technique of Loop-mediated isothermal DNA amplification (LAMP) through the 3MTM Molecular Detection Assay 2: Salmonella (MDS®), in accordance with Ordinance number 126 of the Ministry of Agriculture, for the detection of Salmonella sp. in drag swab. The methodology followed ISO 16140-2: 2016, with the analysis naturally contaminated drag swab samples collected from broiler aviaries and artificially contaminated with salmonella ATCCs. Of the 300 samples processed in protocol A (pre-enrichment tetrathionate broth (TT)), 45 were positive for Salmonella sp., 242 negative, one false-positive, and 12 false-negative, while of the 300 samples analyzed in protocol B (pre-enrichment brain-heart infusion broth (BHI)), 40 were positive, 256 negative, one false-positive, and three false-negative. The result for protocol A was a sensitivity of 79%, specificity of 99.6%, Positive Predictive Value (PPV) of 98%, and Negative Predictive Value (NPV) of 95%; and for protocol B, 93% sensitivity, 99.6% specificity, 98% PPV, and 99% NPV. Both protocols were associated with the reference method (p>0.05), concluding that the MDS® can be used for the qualitative detection of Salmonella sp.

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.

Dietary α-linolenic acid supplementation enhances resistance to Salmonella Typhimurium challenge in chickens by altering the intestinal mucosal barrier integrity and cecal microbes.

Salmonella is an important foodborne pathogen. Given the ban on the use of antibiotics during the egg-laying period in China, finding safe and effective alternatives to antibiotics to reduce Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) infections in chickens is essential for the prevention and control of this pathogen and the protection of human health. Numerous studies have shown that unsaturated fatty acids have a positive effect on intestinal inflammation and resistance to infection by intestinal pathogens. Here we investigated the protective effect of α-linolenic acid (ALA) against S. Typhimurium infection in chickens and further explored its mechanism of action. We added different proportions of ALA to the feed and observed the effect of ALA on S. Typhimurium colonization using metagenomic sequencing technology and physiological index measurements. The role of gut flora on S. Typhimurium colonization was subsequently verified by fecal microbiota transplantation (FMT). We found that ALA protects chickens from S. Typhimurium infection by reducing intestinal inflammation through remodeling the gut microbiota, up-regulating the expression of ileocecal barrier-related genes, and maintaining the integrity of the intestinal epithelium. Our data suggest that supplementation of feed with ALA may be an effective strategy to alleviate S. Typhimurium infection in chickens.

Characterization of Salmonella species from poultry slaughterhouses in South Korea: carry-over transmission of Salmonella Thompson ST292 in slaughtering process.

IMPORTANCE: Salmonella outbreaks linked to poultry meat have been reported continuously worldwide. Therefore, Salmonella contamination of poultry meats in slaughterhouses is one of the critical control points for reducing disease outbreaks in humans. OBJECTIVE: This study examined the carry-over contamination of Salmonella species through the entire slaughtering process in South Korea. METHODS: From 2018 to 2019, 1,097 samples were collected from the nine slaughterhouses distributed nationwide. One hundred and seventeen isolates of Salmonella species were identified using the invA gene-specific polymerase chain reaction, as described previously. The serotype, phylogeny, and antimicrobial resistance of isolates were examined. RESULTS: Among the 117 isolates, 93 were serotyped into Salmonella Mbandaka (n = 36 isolates, 30.8%), Salmonella Thompson (n = 33, 28.2%), and Salmonella Infantis (n = 24, 20.5%). Interestingly, allelic profiling showed that all S. Mbandaka isolates belonged to the lineage of the sequence type (ST) 413, whereas all S. Thompson isolates were ST292. Moreover, almost all S. Thompson isolates (97.0%, 32/33 isolates) belonging to ST292 were multidrug-resistant and possessed the major virulence genes whose products are required for full virulence. Both serotypes were distributed widely throughout the slaughtering process. Pulsed-field gel electrophoretic analysis demonstrated that seven S. Infantis showed 100% identities in their phylogenetic relatedness, indicating that they were sequentially transmitted along the slaughtering processes. CONCLUSIONS AND RELEVANCE: This study provides more evidence of the carry-over transmission of Salmonella species during the slaughtering processes. ST292 S. Thompson is a potential pathogenic clone of Salmonella species possibly associated with foodborne outbreaks in South Korea.

Chicken intestinal organoids: a novel method to measure the mode of action of feed additives.

There is a rapidly growing interest in how the avian intestine is affected by dietary components and feed additives. The paucity of physiologically relevant models has limited research in this field of poultry gut health and led to an over-reliance on the use of live birds for experiments. The development of complex 3D intestinal organoids or "mini-guts" has created ample opportunities for poultry research in this field. A major advantage of the floating chicken intestinal organoids is the combination of a complex cell system with an easily accessible apical-out orientation grown in a simple culture medium without an extracellular matrix. The objective was to investigate the impact of a commercial proprietary blend of organic acids and essential oils (OA+EO) on the innate immune responses and kinome of chicken intestinal organoids in a Salmonella challenge model. To mimic the in vivo prolonged exposure of the intestine to the product, the intestinal organoids were treated for 2 days with 0.5 or 0.25 mg/mL OA+EO and either uninfected or infected with Salmonella and bacterial load in the organoids was quantified at 3 hours post infection. The bacteria were also treated with OA+EO for 1 day prior to challenge of the organoids to mimic intestinal exposure. The treatment of the organoids with OA+EO resulted in a significant decrease in the bacterial load compared to untreated infected organoids. The expression of 88 innate immune genes was investigated using a high throughput qPCR array, measuring the expression of 88 innate immune genes. Salmonella invasion of the untreated intestinal organoids resulted in a significant increase in the expression of inflammatory cytokine and chemokines as well as genes involved in intracellular signaling. In contrast, when the organoids were treated with OA+EO and challenged with Salmonella, the inflammatory responses were significantly downregulated. The kinome array data suggested decreased phosphorylation elicited by the OA+EO with Salmonella in agreement with the gene expression data sets. This study demonstrates that the in vitro chicken intestinal organoids are a new tool to measure the effect of the feed additives in a bacterial challenge model by measuring innate immune and protein kinases responses.

Internal Organ Colonization by Salmonella Enteritidis in Layer Pullets Infected at Two Different Ages During Rearing in Cage-Free Housing.

The poultry-housing environment plays a significant role in the transmission and persistence of the egg-associated pathogen Salmonella Enteritidis in laying flocks. The commercial egg industry is in the midst of a transition toward cage-free housing, but the food safety ramifications of this shift are not yet certain. The present study assessed internal organ colonization by Salmonella Enteritidis in layer pullets reared in cage-free housing and infected at two different ages. Groups of 280 pullets were transferred from the rearing facility (at 9 wk of age in one trial and 15 wk in another) to a containment facility with four isolation rooms simulating commercial cage-free barns with perches and nest boxes (70 birds/room). Twenty-four pullets in each room were orally inoculated with Salmonella Enteritidis immediately after placement in the containment facility. At 1-2 wk postinoculation in each trial, samples of liver, spleen, and intestinal tract were collected from all birds in two rooms for bacteriologic culturing to detect Salmonella Enteritidis. At 21-22 wk of age, samples of spleen, ovary, and intestinal tract were similarly collected and tested from all birds in the remaining two rooms. Among samples collected at 1-2 wk postinoculation, Salmonella Enteritidis was isolated significantly more often from groups of pullets infected initially at 15 wk of age than from those infected at 9 wk (61% vs. 38% of livers, 59% vs. 31% of spleens, and 84% vs. 57% of intestines). Among samples collected at 21-22 wk of age, the frequency of recovery of Salmonella Enteritidis was again significantly greater in birds infected at 15 wk of age than in those infected at 9 wk (16% vs. 6% of spleens, 9% vs. 1% of ovaries, and 26% vs. 10% of intestines). These data suggest that Salmonella Enteritidis infections introduced into flocks during the later stages of pullet rearing have greater potential to persist into the early phase of egg production.

Nota de investigación- Colonización de órganos internos por Salmonella Enteritidis en pollitas de postura infectadas en dos edades diferentes durante la crianza en alojamiento sin jaulas. El ambiente en alojamientos avícolas juega un papel importante en la transmisión y persistencia del patógeno asociado a los huevos Salmonella Enteritidis en parvadas postura. La industria comercial del huevo se encuentra en medio de una transición hacia alojamientos sin jaulas, pero las ramificaciones de este cambio en la seguridad alimentaria aún no están determinadas. El presente estudio evaluó la colonización de órganos internos por Salmonella Enteritidis en pollitas de postura criadas en alojamientos sin jaulas e infectadas a dos edades diferentes. Se transfirieron grupos de 280 pollitas desde las instalaciones de cría (a las 9 semanas de edad en un ensayo y a las 15 semanas en un segundo ensayo) a una instalación de contención con cuatro salas de aislamiento que simulaban alojamientos comerciales sin jaulas con perchas y nidos (70 aves/sala). Veinticuatro pollitas en cada sala fueron inoculadas oralmente con Salmonella Enteritidis inmediatamente después de su colocación en la instalación de contención. En cada ensayo, de una a dos semanas después de la inoculación, se recolectaron muestras de hígado, bazo y tracto intestinal para cultivo bacteriológico de todas las aves en dos salas para detectar Salmonella Enteritidis. A las 21-22 semanas de edad, se recolectaron y analizaron de manera similar muestras de bazo, ovario y tracto intestinal de todas las aves en las dos salas restantes. Entre las muestras recolectadas entre una y dos semanas después de la inoculación, Salmonella Enteritidis se aisló significativamente con mayor frecuencia en grupos de pollitas infectadas inicialmente a las 15 semanas de edad que en aquellas infectadas a las 9 semanas (61% contra 38 % en los hígados, 59% contra 31% de bazos y 84 % contra 57% en intestinos). Entre las muestras recolectadas a las 21-22 semanas de edad, la frecuencia de recuperación de Salmonella Enteritidis fue nuevamente significativamente mayor en aves infectadas a las 15 semanas de edad que en aquellas infectadas a las 9 semanas (16% contra 6% de bazos, 9% contra 1% en ovarios y 26% contra 10% de los intestinos). Estos datos sugieren que las infecciones por Salmonella Enteritidis introducidas en las parvadas durante las últimas etapas de la cría de pollitas tienen un mayor potencial para persistir en la fase inicial de la producción de huevos.

Evaluation of immunogenicity and protective efficacy of outer membrane vesicles from Salmonella Typhimurium and Salmonella Choleraesuis.

Outer membrane vesicles (OMVs) are membranous structures frequently observed in Gram-negative bacteria that contain bioactive substances. These vesicles are rich in bacterial antigens that can activate the host's immune system, making them a promising candidate vaccine to prevent and manage bacterial infections. The aim of this study was to assess the immunogenicity and protective efficacy of OMVs derived from Salmonella enterica serovar Typhimurium and S. Choleraesuis, while also focusing on enhancing OMV production. Initial experiments showed that OMVs from wild-type strains did not provide complete protection against homologous Salmonella challenge, possible due to the presence of flagella in the purified OMVs samples, which may elicit an unnecessary immune response. To address this, flagellin-deficient mutants of S. Typhimurium and S. Choleraesuis were constructed, designated rSC0196 and rSC0199, respectively. These mutants exhibited reduced cell motility and their OMVs were found to be flagellin-free. Immunization with non-flagellin OMVs derived from rSC0196 induced robust antibody responses and improved survival rates in mice, as compared to the OMVs derived from the wild-type UK-1. In order to enhance OMV production, deletions of ompA or tolR were introduced into rSC0196. The deletion of tolR not only increase the yield of OMVs, but also conferred complete protection against homologous S. Typhimurium challenge in mice. Collectively, these findings indicate that the flagellin-deficient OMVs with a tolR mutation have the potential to serve as a versatile vaccine platform, capable of inducing broad-spectrum protection against significant pathogens.

Salmonella carriage and change in serovar distribution in broiler giblets at slaughterhouse level in Turkiye: first report using ISO 6579-1:2017 and ISO 6579-3:2014.

This study aimed to determine the prevalence and serovar distribution of salmonellae in liver, heart, and spleen (LHS) and gizzard (G) of slaughtered broilers. For this, a total of 60 sample units, comprised of 30 LHS and 30 G collected from 3 slaughterhouses, were analysed by reference methods for detection and serotyping as revised ISO 6579-1:2017 and ISO 6579-3:2014, respectively. Also, Salmonella-specific real-time PCR (Salm-PCR) was used for species confirmation, while Salmonella Enteritidis (S. Enteritidis) and Salmonella Typhimurium (S. Typhimurium) specific real-time PCR (SE/ST-PCR) was evaluated to determine its efficiency for rapid detection of the serovars mandated in current legal regulations compared to standard serotyping. All LHS (100%-30/30) and 90% (27/30) of G samples harbored Salmonella with an overall prevalence of 95% (57/60) in samples examined, where all isolates were confirmed as Salmonella by Salm-PCR. The most prevalent serovar in broiler giblets was S. Virchow (80.70%-46/57) followed by S. Enteritidis (19.30%-11/57). SE/ST-PCR (%17.54-10/57) could not detect one G isolate, which was serotyped as S. Enteritidis by standard serotyping. High relative accuracy (98.25%), sensitivity (100%) and specificity (100%), and agreement between methods (κ: 0.94) verified SE/ST-PCR's potential to be used as an alternative in rapid detection of S. Enteritidis and S. Typhimurium. Data on high Salmonella prevalence in broiler giblets of slaughterhouse origin, and detection of the pathogen by the implementation of all requirements indicated in the revised ISO 6579-1:2017 standard method, enabling the determination of actual prevalence in the samples with high sensitivity and specificity is of significance for public health. Additionally, identification of S. Virchow as the dominant serovar followed by S. Enteritidis with a relatively lower prevalence, and absence of S. Typhimurium in broiler giblets are important findings for Turkiye. This up to date data, obtained by strict application of ISO 6579-3:2014 procedures, indicated a shift in circulating serovars in the broiler industry. The objective findings in this study would bring awareness to national/international literature, and may be of use in future improvements in legal regulations.

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.

Characterization, genome analysis, and therapeutic evaluation of a novel Salmonella phage vB_SalS_JNS02: a candidate bacteriophage for phage therapy.

Phage therapy is gaining momentum as an alternative to antibiotics in the treatment of salmonellosis caused by Salmonella. In this study, a novel Salmonella phage, vB_SalS_JNS02, was isolated successfully from poultry farms in Shandong, China. The biological characteristics of vB_SalS_JNS02 were analysed, which revealed a short latent period of approximately 10 min and a burst size of 110 PFU/cell. Moreover, vB_SalS_JNS02 exhibited remarkable stability across a wide pH range (pH 3-12) and temperatures ranging from 30 to 80°C. Genome sequencing analysis provided valuable insights into the genetic composition of vB_SalS_JNS02, which consists of a double-stranded DNA genome that spans 42,450 base pairs and has a G + C content of 49.4%. Of significant importance, the genomic sequence of vB_SalS_JNS02 did not contain any genes related to lysogenicity, virulence, or antibiotic resistance. The phage's efficacy was evaluated in a larval challenge study. Treatment with the phage resulted in increased survival of Galleria mellonella larvae (100, 70, and 85%) (MOI 0.1) in the prophylactic treatment, co-infection treatment, and remedial treatment experiments, respectively. Another in vivo experiment investigated the potential application of the phage in broiler chickens and revealed that a single oral dose of vB_SalS_JNS02 (108 PFU/mL, 100 µL/chick) administered 3 h after S. enteritidis oral administration provided effective protection. The introduction of bacteriophage not only enhances the production of secretory immunoglobulin A (sIgA), but also induces alterations in the composition of the gut microbial community. Phage therapy increases the relative abundance of beneficial bacteria, which helps to maintain intestinal barrier homeostasis. However, it is unable to fully restore the disrupted intestinal microbiome caused by S. enteritidis infection. Importantly, no significant adverse effects were observed in the animal subjects following oral administration of the phage, and our findings highlight vB_SalS_JNS02 is a hopeful candidate as a promising tool to target Salmonella infections in poultry.

Torsion of the caudate lobe of the liver and concurrent necrohemorrhagic typhlocolitis in a zoo-housed Patagonian mara.

Liver lobe torsion has been reported in many species, with frequent reports in rabbits. Here we describe caudate liver lobe torsion and concurrent necrohemorrhagic typhlocolitis in a Patagonian mara (syn: Patagonian cavy, Patagonian hare, Dolichotis patagonum). Following acute death, postmortem examination findings included torsion of the hepatic caudate process, which had fibrous adhesions to the pancreas indicating chronicity. The cecal apex and proximal 30 cm of colon had regionally reddened serosa and diffusely roughened and reddened mucosa with brown-red and granular luminal contents. Key histologic findings included massive necrosis of the torsed hepatic caudate lobe, consistent with infarction, necrotizing hepatitis in remaining areas of liver, necrohemorrhagic typhlocolitis, adrenocortical necrosis and hemorrhage, and renal tubular degeneration and necrosis with tubular casts. Bacterial culture of cecal contents yielded pure growth of Salmonella spp. Death was attributed to toxemia or bacteremia resulting from Salmonella spp. infection, as the hepatic lobe torsion appeared chronic. It was undetermined if the liver lobe torsion predisposed to gastrointestinal compromise and infection. Patagonian maras have some anatomical similarities to rabbits and are highly cursorial, not dissimilar to hares, Lepus spp. We speculate that these characteristics may increase the likelihood of hepatic caudate lobe torsion in this species.

Pan-Genome-Wide Association Study reveals a key role of the salmochelin receptor IroN in the biofilm formation of Salmonella Typhimurium and its monophasic variant 4,[5],12:i:.

Salmonella enterica subsp. enterica serovar Typhimurium variant 4,[5],12:i:- (so called S. 4,[5],12:i:-) has rapidly become one of the most prevalent serovars in humans in Europe, with clinical cases associated with foodborne from pork products. The mechanisms, genetic basis and biofilms relevance by which S. 4,[5],12:i:- maintains and spreads its presence in pigs remain unclear. In this study, we examined the genetic basis of biofilm production in 78 strains of S. 4,[5],12:i:- (n = 57) and S. Typhimurium (n = 21), from human gastroenteritis, food products and asymptomatic pigs. The former showed a lower Specific Biofilm Formation index (SBF) and distant phylogenetic clades, suggesting that the ability to form biofilms is not a crucial adaptation for the S. 4,[5],12:i:- emerging success in pigs. However, using a pan-Genome-Wide Association Study (pan-GWAS) we identified genetic determinants of biofilm formation, revealing 167 common orthologous groups and genes associated with the SBF. The analysis of annotated sequences highlighted specific genetic deletions in three chromosomal regions of S. 4,[5],12:i:- correlating with SBF values: i) the complete fimbrial operon stbABCDE widely recognized as the most critical factor involved in Salmonella adherence; ii) the hxlA, hlxB, and pgiA genes, which expression in S. Typhimurium is induced in the tonsils during swine infection, and iii) the entire iroA locus related to the characteristic deletion of the second-phase flagellar genomic region in S. 4,[5],12:i:-. Consequently, we further investigated the role of the iro-genes on biofilm by constructing S. Typhimurium deletion mutants in iroBCDE and iroN. While iroBCDE showed no significant impact, iroN clearly contributed to S. Typhimurium biofilm formation. In conclusion, the pan-GWAS approach allowed us to uncover complex interactions between genetic and phenotypic factors influencing biofilm formation in S. 4,[5],12:i:- and S. Typhimurium.

Research Note: Validation of a new differentiation approach using the commercial ASAPTM media to detect the Salmonella 441/014 vaccine strain.

Vaccination is one of the most important control tools to reduce Salmonella in poultry production. In order for a live vaccine to be licensed for field use it should be provided with the detection methods to differentiate it from field strains. This paper aims to describe the validation of an alternative method for the differentiation of the Salmonella 441/014 vaccine strain from field strains, using a chromogenic Media, ASAP from bioMérieux. The ASAP-based differentiation method was compared with already authorized methods, namely the Anicon SE Kylt PCR DIVA 1 assay and Ceva S-Check Salmonella differentiation kit, following the ISO 16140-6:2019 validation method guidelines. A Generalised Linear Model was fitted to the data to determine the inclusivity and exclusivity of differentiation methods (PCR Kylt vs. S-Check vs. ASAPTM). Statistical differences were based on a P-value level of < 0.05 (SPSS Inc., Chicago, IL). In this study, we show that the ASAP media was able to differentiate Salmonella Enteritidis vaccine strains from field strains, obtaining 100% agreement between the three differentiation assays. This differentiation approach is quicker, easier to deploy and cheaper as compared to alternative methods.

Prevalence and antimicrobial susceptibility of Salmonella enteritidis and Salmonella typhimurium isolated from hen eggs and quail eggs in Karaj, Iran.

BACKGROUND AND AIM: Different Salmonella serotypes are considered one of the most important food pathogens in the world. Poultry meat and eggs are the primary carriers of Salmonella in human populations. This study aimed to estimate the Salmonella enteritidis and Salmonella typhimurium contamination rates of retail hen and quail eggs in Karaj, Iran. Moreover, the antimicrobial resistance patterns of the strains were evaluated, and the efficiency of the standard culture method and multiplex polymerase chain reaction (m-PCR) were compared. MATERIALS AND METHODS: In this descriptive cross-sectional study over 1 year (Jan-Dec 2022), 150 commercial and 150 backyard hen eggs and 300 commercial quail eggs, without cracks and fractures, were collected randomly from best selling groceries in Karaj city. All samples were examined for Salmonella contamination independently by standard culture and m-PCR approaches. A standard disc diffusion method was employed to assess the antimicrobial susceptibility of the strains against 18 antimicrobial agents. RESULTS: Out of 300 examined eggs, 2 S. enteritidis strains were isolated from the shell of backyard hen eggs. The same serotype was also detected in the contents of one of these two eggs. One S. typhimurium was isolated from the shell of a commercial hen egg. Overall, the Salmonella contamination of the shell and contents was 1% and 0.3%, respectively. Salmonella was not isolated from the eggshells or the contents of the quail eggs. There was complete agreement between the results of m-PCR and the standard culture methods. Among the 18 tested antibiotics, the highest resistance was recorded for colistin (100%), followed by nalidixic acid (75%). CONCLUSION: As most Salmonella spp. are associated with human food poisoning, continuous surveillance is required to effectively reduce the risk posed by contaminated poultry eggs. Furthermore, mandatory monitoring of antimicrobial use on Iranian poultry farms is recommended.

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.

Prevalence, antimicrobial resistance and detection of virulence genes of Escherichia coli and Salmonella spp. isolated from white-lipped peccaries and collared peccaries.

Salmonella spp. and Escherichia coli are implicated in human and animal infections and require antimicrobial treatment in many situations. Faecal samples of healthy white-lipped peccaries (Pecari tajacu) (n = 30) and collared peccaries (Tayassu pecari ) (n = 60) obtained in three farms located in the Midwest Brazil. The antimicrobial profiles of commensal E. coli from P. tajacu and T. pecari from commercial herds in Brazil were isolated and analyzed and virulence genes were detected. Among 90 healthy animals, no Salmonella spp. were isolated. However, 30 samples (27%) tested positive for E. coli, with 18 isolates from P. tajacu and 12 from T. pecari, representing frequencies of 58.0% and 38.7%, respectively. Additionally, other Enterobacteriaceae family bacteria were detected but not included in this analysis. However, individual samples from 30 animals tested positive for E. coli, of which 16 were isolated from P. tajacu presenting multidrug resistance and six were isolated from T. pecari presenting a similar pattern. The E. coli virulence genes detected were papC (pilus-associated pyelonephritis) in five isolates, tsh (temperature-sensitive hemagglutinin) in one isolate, and eae (enteric attachment and effacement) in one isolate. The serum resistance gene, iss (increased serum survival), was detected in four isolates. An association between these genes and the presence of hemolysin was also observed in one isolate. Thus, T. pecari and P. tajacu are potential reservoirs of pathogenic and multidrug-resistant and E. coli. Faecal E. coli of healthy P. tajacu and T. pecari could act as a possible reservoir of antimicrobial resistance genes in environment.

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.

Battling Salmonella enteritidis infections: integrating proteomics and in vivo assessment of Galla Chinensis tannic acid.

Salmonella infections pose a significant threat to animal and human health. Phytochemicals present a potential alternative treatment. Galla chinensis tannic acid (GCTA), a hydrolyzable polyphenolic compound, inhibits bacterial growth and demonstrates potential as an alternative or supplement to antibiotics to prevent Salmonella infections. However, little is known about the antimicrobial mechanism of GCTA against Salmonella. Here, we revealed 456 differentially expressed proteins upon GCTA treatment, impacting pathways related to DNA replication, repair, genomic stability, cell wall biogenesis, and lipid metabolism using TMT-labeled proteomic analysis. TEM analysis suggested altered bacterial morphology and structure post-treatment. A Salmonella-infected-mouse model indicated that GCTA administration improved inflammatory markers, alleviated intestinal histopathological alterations, and reduced Salmonella enterica serovar Enteritidis (S. Enteritidis) colonization in the liver and spleen of Salmonella-infected mice. The LD50 of GCTA was 4100 mg/kg with an oral single dose, vastly exceeding the therapeutic dose. Thus, GCTA exhibited antibacterial and anti-infective activity against S. Enteritidis. Our results provided insight into the molecular mechanisms of these antibacterial effects, and highlights the potential of GCTA as an alternative to antibiotics.

Lipopolysaccharide with long O-antigen is crucial for Salmonella Enteritidis to evade complement activity and to facilitate bacterial survival in vivo in the Galleria mellonella infection model.

Bacterial resistance to serum is a key virulence factor for the development of systemic infections. The amount of lipopolysaccharide (LPS) and the O-antigen chain length distribution on the outer membrane, predispose Salmonella to escape complement-mediated killing. In Salmonella enterica serovar Enteritidis (S. Enteritidis) a modal distribution of the LPS O-antigen length can be observed. It is characterized by the presence of distinct fractions: low molecular weight LPS, long LPS and very long LPS. In the present work, we investigated the effect of the O-antigen modal length composition of LPS molecules on the surface of S. Enteritidis cells on its ability to evade host complement responses. Therefore, we examined systematically, by using specific deletion mutants, roles of different O-antigen fractions in complement evasion. We developed a method to analyze the average LPS lengths and investigated the interaction of the bacteria and isolated LPS molecules with complement components. Additionally, we assessed the aspect of LPS O-antigen chain length distribution in S. Enteritidis virulence in vivo in the Galleria mellonella infection model. The obtained results of the measurements of the average LPS length confirmed that the method is suitable for measuring the average LPS length in bacterial cells as well as isolated LPS molecules and allows the comparison between strains. In contrast to earlier studies we have used much more precise methodology to assess the LPS molecules average length and modal distribution, also conducted more subtle analysis of complement system activation by lipopolysaccharides of various molecular mass. Data obtained in the complement activation assays clearly demonstrated that S. Enteritidis bacteria require LPS with long O-antigen to resist the complement system and to survive in the G. mellonella infection model.