Salmonella exploits membrane reservoirs for invasion of host cells.

Salmonella utilizes a type 3 secretion system to translocate virulence proteins (effectors) into host cells during infection1. The effectors modulate host cell machinery to drive uptake of the bacteria into vacuoles, where they can establish an intracellular replicative niche. A remarkable feature of Salmonella invasion is the formation of actin-rich protuberances (ruffles) on the host cell surface that contribute to bacterial uptake. However, the membrane source for ruffle formation and how these bacteria regulate membrane mobilization within host cells remains unclear. Here, we show that Salmonella exploits membrane reservoirs for the generation of invasion ruffles. The reservoirs are pre-existing tubular compartments associated with the plasma membrane (PM) and are formed through the activity of RAB10 GTPase. Under normal growth conditions, membrane reservoirs contribute to PM homeostasis and are preloaded with the exocyst subunit EXOC2. During Salmonella invasion, the bacterial effectors SipC, SopE2, and SopB recruit exocyst subunits from membrane reservoirs and other cellular compartments, thereby allowing exocyst complex assembly and membrane delivery required for bacterial uptake. Our findings reveal an important role for RAB10 in the establishment of membrane reservoirs and the mechanisms by which Salmonella can exploit these compartments during host cell invasion.

Genetic Characteristics of Salmonella Isolates Recovered From Reused Broiler Litter Over Three Successive Flocks.

Salmonella infections are a leading cause of bacterial food-borne illness worldwide. Infections are highly associated with the consumption of contaminated food, and in particular, chicken meat. The severity of Salmonella infections depends on the presence of antimicrobial resistance genes and virulence factors. While there are many studies which have investigated Salmonella strains isolated from postharvest chicken samples, there is a gap in our understanding of the genetic properties that influence the persistence of Salmonella in preharvest and in particular their makeup of antimicrobial resistance genes and virulence factors. We used whole genome sequencing and hierarchical clustering to characterize and classify the genetic diversity of Salmonella enterica isolates (n = 55) recovered from the litter of commercial broiler chicken raised in four colocated broiler houses of one integrated farm over three consecutive flocks. The chicken were raised under a newly adopted "No Antibiotics Ever" program, and copper sulfate was administered via drinking water. In-silico serovar prediction identified three S. enterica serovars: Enteritidis (n = 12), Kentucky (n = 40), and Senftenberg (n = 3). Antimicrobial susceptibility testing revealed that only one S. Kentucky isolate was resistant to streptomycin, while the remaining isolates were susceptible to all antibiotics tested. Metal resistance operons, including copper and silver, were identified chromosomally and on plasmids in serovar Senftenberg and Kentucky isolates, respectively, while serovar Enteritidis carried several virulence factors on plasmids. Serovar Kentucky isolates harboring metal resistance operons were the only Salmonella isolates recovered from the litter of third flock cohort. These results suggest that there might be environmental selection for Salmonella strains carrying plasmid-associated metal resistance and virulence genes, which could play a role in their persistence in litter.

Persistent Salmonella infections in humans are associated with mutations in the BarA/SirA regulatory pathway.

Several bacterial pathogens, including Salmonella enterica, can cause persistent infections in humans by mechanisms that are poorly understood. By comparing genomes of isolates longitudinally collected from 256 prolonged salmonellosis patients, we identified repeated mutations in global regulators, including the barA/sirA two-component regulatory system, across multiple patients and Salmonella serovars. Comparative RNA-seq analysis revealed that distinct mutations in barA/sirA led to diminished expression of Salmonella pathogenicity islands 1 and 4 genes, which are required for Salmonella invasion and enteritis. Moreover, barA/sirA mutants were attenuated in an acute salmonellosis mouse model and induced weaker transcription of host immune responses. In contrast, in a persistent infection mouse model, these mutants exhibited long-term colonization and prolonged shedding. Taken together, these findings suggest that selection of mutations in global virulence regulators facilitates persistent Salmonella infection in humans, by attenuating Salmonella virulence and inducing a weaker host inflammatory response.

A two-step activation mechanism enables mast cells to differentiate their response between extracellular and invasive enterobacterial infection.

Mast cells localize to mucosal tissues and contribute to innate immune defense against infection. How mast cells sense, differentiate between, and respond to bacterial pathogens remains a topic of ongoing debate. Using the prototype enteropathogen Salmonella Typhimurium (S.Tm) and other related enterobacteria, here we show that mast cells can regulate their cytokine secretion response to distinguish between extracellular and invasive bacterial infection. Tissue-invasive S.Tm and mast cells colocalize in the mouse gut during acute Salmonella infection. Toll-like Receptor 4 (TLR4) sensing of extracellular S.Tm, or pure lipopolysaccharide, causes a modest induction of cytokine transcripts and proteins, including IL-6, IL-13, and TNF. By contrast, type-III-secretion-system-1 (TTSS-1)-dependent S.Tm invasion of both mouse and human mast cells triggers rapid and potent inflammatory gene expression and >100-fold elevated cytokine secretion. The S.Tm TTSS-1 effectors SopB, SopE, and SopE2 here elicit a second activation signal, including Akt phosphorylation downstream of effector translocation, which combines with TLR activation to drive the full-blown mast cell response. Supernatants from S.Tm-infected mast cells boost macrophage survival and maturation from bone-marrow progenitors. Taken together, this study shows that mast cells can differentiate between extracellular and host-cell invasive enterobacteria via a two-step activation mechanism and tune their inflammatory output accordingly.

Diversity of evolution in MDR monophasic S. Typhimurium among food animals and food products in Southern China from 2011 to 2018.

The monophasic variant of Salmonella enterica serovar Typhimurium with the antigenic formula 1,4,[5],12:i:- is one of the most common pathogenic bacteria causing global food-borne outbreaks. However, the research on molecular characteristics and evolution of monophasic S. typhimurium in China is still lacking. In the current study, 59 monophasic S. typhimurium strains were isolated from food animals and food products in South China between 2011 and 2018. A total of 87.5 % of monophasic S. typhimurium isolates were grouped into one independent clade with other monophasic S. typhimurium strains in China distinct from other countries by phylogenomic analysis. These isolates possess variable genotypes, including multiple ARGs on plasmid IncHI2, diverse evolutions at the fljAB locus, and virulence factors. Our results suggest that the monophasic S. typhimurium isolates currently circulating in China might be an independent epidemic subtype.

Fisetin inhibits Salmonella Typhimurium type III secretion system regulator HilD and reduces pathology in vivo.

IMPORTANCE: Salmonella spp. remains a major worldwide health concern that causes significant morbidity and mortality in both humans and animals. The spread of antimicrobial resistant strains has declined the efficacy of conventional chemotherapy. Thus, novel anti-infection drugs or strategies are needed. Anti-virulence strategy represents one of the promising means for the treatment of bacterial infections. In this study, we found that the natural compound fisetin could inhibit Salmonella invasion of host cells by targeting SPI-1 regulation. Fisetin treatment impaired the interaction of the regulatory protein HilD with the promoters of its target genes, thereby suppressing the expression of T3SS-1 effectors as well as structural proteins. Moreover, fisetin treatment could reduce pathology in the Salmonella murine infection model. Collectively, our results suggest that fisetin may serve as a promising lead compound for the development of anti-Salmonella drugs.

Microbiome diversity protects against pathogens by nutrient blocking.

The human gut microbiome plays an important role in resisting colonization of the host by pathogens, but we lack the ability to predict which communities will be protective. We studied how human gut bacteria influence colonization of two major bacterial pathogens, both in vitro and in gnotobiotic mice. Whereas single species alone had negligible effects, colonization resistance greatly increased with community diversity. Moreover, this community-level resistance rested critically upon certain species being present. We explained these ecological patterns through the collective ability of resistant communities to consume nutrients that overlap with those used by the pathogen. Furthermore, we applied our findings to successfully predict communities that resist a novel target strain. Our work provides a reason why microbiome diversity is beneficial and suggests a route for the rational design of pathogen-resistant communities.

Self-assembly of H2S-responsive nanoprodrugs based on natural rhein and geraniol for targeted therapy against Salmonella Typhimurium.

Salmonellosis is a globally extensive food-borne disease, which threatens public health and results in huge economic losses in the world annually. The rising prevalence of antibiotic resistance in Salmonella poses a significant global concern, emphasizing an imperative to identify novel therapeutic agents or methodologies to effectively combat this predicament. In this study, self-assembly hydrogen sulfide (H2S)-responsive nanoprodrugs were fabricated with poly(α-lipoic acid)-polyethylene glycol grafted rhein and geraniol (PPRG), self-assembled into core-shell nanoparticles via electrostatic, hydrophilic and hydrophobic interactions, with hydrophilic exterior and hydrophobic interior. The rhein and geraniol are released from self-assembly nanoprodrugs PPRG in response to Salmonella infection, which is known to produce hydrogen sulfide (H2S). PPRG demonstrated stronger antibacterial activity against Salmonella compared with rhein or geraniol alone in vitro and in vivo. Additionally, PPRG was also able to suppress the inflammation and modulate gut microbiota homeostasis. In conclusion, the as-prepared self-assembly nanoprodrug sheds new light on the design of natural product active ingredients and provides new ideas for exploring targeted therapies for specific Enteropathogens. Graphical  illustration for construction of self-assembly nanoprodrugs PPRG and its antibacterial and anti-inflammatory activities on experimental Salmonella infection in mice.

The Impact of Substitutions at Positions 1 and 8 of Fluoroquinolones on the Activity Against Mutant DNA Gyrases of Salmonella Typhimurium.

Although many drug-resistant nontyphoidal Salmonella (NTS) infections are reported globally, their treatment is challenging owing to the ineffectiveness of the currently available antimicrobial drugs against resistant bacteria. It is therefore essential to discover novel antimicrobial drugs for the management of these infections. In this study, we report high inhibitory activities of the novel fluoroquinolones (FQs; WQ-3810 and WQ-3334) with substitutions at positions R-1 by 6-amino-3,5-difluoropyridine-2-yl and R-8 by methyl group or bromine, respectively, against wild-type and mutant DNA gyrases of Salmonella Typhimurium. The inhibitory activities of these FQs were assessed against seven amino acid substitutions in DNA gyrases conferring FQ resistance to S. Typhimurium, including high-level resistant mutants, Ser83Ile and Ser83Phe-Asp87Asn by in vitro DNA supercoiling assay. Drug concentrations of WQ compounds with 6-amino-3,5-difluoropyridine-2-yl that suppressed DNA supercoiling by 50% (IC50) were found to be ∼150-fold lower than ciprofloxacin against DNA gyrase with double amino acid substitutions. Our findings highlight the importance of the chemical structure of an FQ drug on its antimicrobial activity. Particularly, the presence of 6-amino-3,5-difluoropyridine-2-yl at R-1 and either methyl group or bromine at R-8 of WQ-3810 and WQ-3334, respectively, was associated with improved antimicrobial activity. Therefore, WQ-3810 and WQ-3334 are promising candidates for use in the treatment of patients infected by FQ-resistant Salmonella spp.

HilE represses the activity of the Salmonella virulence regulator HilD via a mechanism distinct from that of intestinal long-chain fatty acids.

The expression of virulence factors essential for the invasion of host cells by Salmonella enterica is tightly controlled by a network of transcription regulators. The AraC/XylS transcription factor HilD is the main integration point of environmental signals into this regulatory network, with many factors affecting HilD activity. Long-chain fatty acids, which are highly abundant throughout the host intestine, directly bind to and repress HilD, acting as environmental cues to coordinate virulence gene expression. The regulatory protein HilE also negatively regulates HilD activity, through a protein-protein interaction. Both of these regulators inhibit HilD dimerization, preventing HilD from binding to target DNA. We investigated the structural basis of these mechanisms of HilD repression. Long-chain fatty acids bind to a conserved pocket in HilD, in a comparable manner to that reported for other AraC/XylS regulators, whereas HilE forms a stable heterodimer with HilD by binding to the HilD dimerization interface. Our results highlight two distinct, mutually exclusive mechanisms by which HilD activity is repressed, which could be exploited for the development of new antivirulence leads.

Invasive non-typhoidal Salmonella (iNTS) aminoglycoside-resistant ST313 isolates feature unique pathogenic mechanisms to reach the bloodstream.

Invasive non-typhoidal Salmonella (iNTS) from the clonal type ST313 (S. Typhimurium ST313) is the primary cause of invasive salmonellosis in Africa. Recently, in Brazil, iNTS ST313 strains have been isolated from different sources, but there is a lack of understanding of the mechanisms behind how these gut bacteria can break the gut barrier and reach the patient's bloodstream. Here, we compare 13 strains of S. Typhimurium ST313, previously unreported isolates, from human blood cultures, investigating aspects of virulence and mechanisms of resistance. Initially, RNAseq analyses between ST13-blood isolate and SL1344 (ST19) prototype revealed 15 upregulated genes directly related to cellular invasion and replication, such as sopD2, sifB, and pipB. Limited information is available about S. Typhimurium ST313 pathogenesis and epidemiology, especially related to the global distribution of strains. Herein, the correlation of strains isolated from different sources in Brazil was employed to compare clinical and non-clinical isolates, a total of 22 genomes were studied by single nucleotide polymorphism (SNPs). The epidemiological analysis of 22 genomes of S. Typhimurium ST313 strains grouped them into three distinct clusters (A, B, and C) by SNP analysis, where cluster A comprised five, group B six, and group C 11. The 13 clinical blood isolates were all resistant to streptomycin, 92.3% of strains were resistant to ampicillin and 15.39% were resistant to kanamycin. The resistance genes acrA, acrB, mdtK, emrB, emrR, mdsA, and mdsB related to the production of efflux pumps were detected in all (100%) strains studied, similar to pathogenic traits investigated. In conclusion, we evidenced that S. Typhimurium ST313 strains isolated in Brazil have unique epidemiology. The elevated frequencies of virulence genes such as sseJ, sopD2, and pipB are a major concern in these Brazilian isolates, showing a higher pathogenic potential.

Clinical and microbiological characterization of Salmonella spp. isolates from patients treated in a university hospital in South America between 2012-2021: a cohort study.

BACKGROUND: Salmonellosis is a major cause of morbidity and mortality and one of the most frequent etiologies of diarrhea in the world. Mortality due to Salmonellosis in Latin America still poorly understood, and there is a lack of studies that evaluate resistance and clinical manifestations. The aims of this study were to characterize patients infected with Salmonella spp. seen in a university hospital in Colombia between 2012 and 2021, to evaluate trends in antibiotic resistance and to determine the proportion of overall mortality and related factors. METHODS: Retrospective observational study. All patients with microbiological diagnosis of Salmonella spp. were included. The sociodemographic, clinical and microbiological characteristics were described, and the proportion of antibiotic resistant isolates per year was estimated. The prevalence of mortality according to age groups was calculated. Log binomial regression models were used to establish factors associated with mortality. RESULTS: Five hundred twenty-two patients were analyzed. Salmonellosis accounted for 0.01% of all medical consultations. The median age was 16 years old. The most common clinical presentation was gastroenteric syndrome (77.1%) and symptoms included diarrhea (79.1%), fever (66.7%), abdominal pain (39.6%) and vomiting (35.2%). Of the Salmonella spp. isolates, 78.2% were not classified, 19.1% corresponded to non-typhoidal Salmonella and 2.7% to Salmonella typhi. Mortality occurs in 4.02% of the patients and was higher in patients with hematologic malignancy (11.6%). When analyzing by age group, the proportion of deaths was 2.8% in patients aged 15 years or younger, while in those older than 15 years it was 5.4%. Factors associated to mortality where bacteremia (aPR = 3.41 CI95%: 1.08-10.76) and to require treatment in the ICU (aPR = 8.13 CI95%: 1.82-37.76). In the last 10 years there has been a steady increase in resistance rates to ciprofloxacin, ampicillin, ampicillin/sulbactam and ceftriaxone, reaching rates above 60% in recent years. CONCLUSIONS: Despite improved availability of antibiotics for the treatment of salmonellosis in the past decades, mortality due to salmonellosis continues occurring in children and adults, mainly in patients with hematological malignancies and bacteremia. Antibiotic resistance rates have increased significantly over the last 10 years. Public health strategies for the control of this disease should be strengthened, especially in vulnerable populations.

RhoB promotes Salmonella survival by regulating autophagy.

Salmonella enterica serovar Typhimurium manipulates cellular Rho GTPases for host cell invasion by effector protein translocation via the Type III Secretion System (T3SS). The two Guanine nucleotide exchange (GEF) mimicking factors SopE and -E2 and the inositol phosphate phosphatase (PiPase) SopB activate the Rho GTPases Rac1, Cdc42 and RhoA, thereby mediating bacterial invasion. S. Typhimurium lacking these three effector proteins are largely invasion-defective. Type III secretion is crucial for both early and later phases of the intracellular life of S. Typhimurium. Here we investigated whether and how the small GTPase RhoB, known to localize on endomembrane vesicles and at the invasion site of S. Typhimurium, contributes to bacterial invasion and to subsequent steps relevant for S. Typhimurium lifestyle. We show that RhoB is significantly upregulated within hours of Salmonella infection. This effect depends on the presence of the bacterial effector SopB, but does not require its phosphatase activity. Our data reveal that SopB and RhoB bind to each other, and that RhoB localizes on early phagosomes of intracellular S. Typhimurium. Whereas both SopB and RhoB promote intracellular survival of Salmonella, RhoB is specifically required for Salmonella-induced upregulation of autophagy. Finally, in the absence of RhoB, vacuolar escape and cytosolic hyper-replication of S. Typhimurium is diminished. Our findings thus uncover a role for RhoB in Salmonella-induced autophagy, which supports intracellular survival of the bacterium and is promoted through a positive feedback loop by the Salmonella effector SopB.

[Clinical features and antimicrobial resistance of invasive non-typhoid Salmonella infection in children at Xiamen].

Objective: To investigate the clinical characteristics, serogroups and antimicrobial resistance of invasive non-typhoid Salmonella infection in children at Xiamen. Methods: Retrospective cohort study. The clinical manifestations, treatment, prognosis, serogroups and antimicrobial resistance of 29 hospitalized children with invasive non-typhoid Salmonella infection confirmed by blood, cerebrospinal fluid, bone marrow and other sterile body fluids or deep pus culture at the Department of Infectious Diseases, the Department of Orthopedics and the Department of General Surgery in Xiamen Children's Hospital from January 2016 to December 2021 were analyzed. According to the clinical diagnosis criteria, the patients were divided into sepsis group and non-sepsis group (bacteremia and local suppurative infection). The inflammatory markers, serogroups distribution and drug resistance were compared between the two groups. Comparison between groups using Mann-Whitney U test and χ2 test. Results: Among the 29 cases, there were 17 males and 12 females, with an onset age of 14 (9, 25) months, and 10 cases (34%) of patients were younger than 1 year old, 15 cases (52%) under 1 to 3 years old, and 4 cases (14%) greater than or equal 3 years old. The onset time of 25 cases (86%) was from April to September. The diseases included 19 cases (66%) septicemia (2 of which were combined with suppurative meningitis), 10 cases (34%) non-sepsis group, including 7 cases bacteremia and 3 cases local suppurative infection (2 cases of osteomyelitis, 1 case of appendicitis with peritonitis). The clinical manifestations were fever in 29 cases (100%), diarrhea and abdominal pain in 18 cases (62%), cough and runny nose in 10 cases (34%). Eighteen cases (62%) were cured and 11 cases (38%) were improved by effective antibiotics treatment. C-reactive protein in sepsis group was significantly higher than that in non-sepsis group (25.2 (16.1, 56.4) vs. 3.4 (0.5, 7.5) mg/L, Z=-3.81, P<0.001).The serogroups of C, B and E were the most prevalent among non-typhoid Salmonella isolates, accounting for 10 cases (34%), 9 cases (31%) and 7 cases (24%) respectively. Antibacterial drug sensitivity test showed that the sensitivity rates of imipenem, ertapenem and piperaciratazobactam were all 100% (31/31), those of ceftazidime, ceftriaxone, and cefepime were 94% (29/31), 94% (29/31) and 97% (30/31) respectively. The drug resistance rates of ampicillin, ampicillin-sulbactam and trimethoprim-sulfamethoxazole were 51% (16/31), 48% (15/31) and 48% (15/31) respectively, those of cefazolin, cefotetan, tobramycin, gentamicin and amikacinwere all 100% (31/31). There were no significant differences in the drug resistance rates of ceftazidime, ceftriaxone, aztreonam, ampicillin-sulbactam, ampicillin, trimethoprim-sulfamethoxazole and ciprofloxacin between the sepsis group and the non-sepsis group (χ2=0.31,0.31,0.00,0.02,0.02,0.02,0.26, all P>0.05). Conclusions: Invasive non-typhoid Salmonella infection in children at Xiamen mainly occurred in infants younger than 3 years old.The main clinical manifestations are fever, abdominal pain and diarrhea. C-reactive protein can be served as the laboratory indicators for indicating sepsis. The third generation of cephalosporins is recommended as the first choice for treatment.

Poultry production as the main reservoir of ciprofloxacin- and tigecycline-resistant extended-spectrum ß-lactamase (ESBL)-producing Salmonella enterica serovar Kentucky ST198.2-2 causing human infections in China.

Salmonella enterica serovar Kentucky (S. Kentucky) has been regarded as a common serotype causing human nontyphoidal salmonellosis, frequently associated with the consumption of contaminated poultry products. Recently, multidrug-resistant (MDR) S. Kentucky ST198 with strong resistance to cefotaxime, ciprofloxacin, and tigecycline has emerged and been frequently detected in both poultry and humans in Europe and Asia. In this study, whole-genome sequencing (WGS) analysis divided 327 S. Kentucky ST198 isolates into two clades, of which ST198.2 is more prevalent than ST198.1 worldwide. We further compared the genomic characteristics of 70 ST198 isolates from animals and humans during 2019-2022 plus previously reported 38 isolates from 2013 to 2019 in China. One hundred five of the 108 isolates were ST198.2, which could be differentiated into two subclades. ST198.2-1 was prevalent in isolates during 2013-2019, while ST198.2-2 has increased to be the predominant subclade in isolates since 2019. CRISPR typing can differentiate the clade ST198.1 isolates from clade ST198.2 ones but cannot differentiate the two subclade isolates. The acquisition of a large multi-drug resistant region in ST198.2-2 enhanced bacterial resistance to ß-lactam, aminoglycoside, amphenicol, and fosfomycin. In addition, compared with the extended-spectrum ß-lactamase (ESBL)-encoding gene blaCTX-M-14b in ST198.2-1, co-existence of blaCTX-M-55 and blaTEM-1B was detected in most of the ST198.2-2 isolates. The emergence of ciprofloxacin- and tigecycline-resistant ESBL-producing S. Kentucky ST198.2-2 strains highlight the necessity for Salmonella surveillance. It is imperative to implement more effective measures to prevent and control transmission of these strains from poultry to humans. IMPORTANCE Salmonella enterica serovar Kentucky (S. Kentucky) can cause human infections through consumption of contaminated food of animal origin, and the emergence of multidrug-resistant (MDR) ST198-S. Kentucky strains are of concern for human and animal health. Based on whole-genome sequencing (WGS) analysis, this study revealed that the clade ST198.2-2 S. Kentucky has increased to the predominant group in both chickens and humans in China since 2019, which is different to previous studies of the prevalent ST198.2-1 S. Kentucky before 2019. Acquirement of a multidrug resistance region (MRR) makes the ST198.2-2 S. Kentucky to be extensively drug-resistant (XDR) isolate compared with ST198.2-1 S. Kentucky. Besides, the ST198.2-2 S. Kentucky was mainly detected in chickens (chicken meat, intestinal contents, and slaughterhouse) and humans, indicating chicken is the main reservoir for these XDR S. Kentucky isolates. Therefore, it is necessary to implement continuous Salmonella surveillance and effective measures, such as the development of phages and novel antibiotics/compounds, to prevent the transmission of XDR ST198.2-2 S. Kentucky from chickens to humans across China.

Parkinson's disease kinase LRRK2 coordinates a cell-intrinsic itaconate-dependent defence pathway against intracellular Salmonella.

Cell-intrinsic defences constitute the first line of defence against intracellular pathogens. The guanosine triphosphatase RAB32 orchestrates one such defence response against the bacterial pathogen Salmonella, through delivery of antimicrobial itaconate. Here we show that the Parkinson's disease-associated leucine-rich repeat kinase 2 (LRRK2) orchestrates this defence response by scaffolding a complex between RAB32 and aconitate decarboxylase 1, which synthesizes itaconate from mitochondrial precursors. Itaconate delivery to Salmonella-containing vacuoles was impaired and Salmonella replication increased in LRRK2-deficient cells. Loss of LRRK2 also restored virulence of a Salmonella mutant defective in neutralizing this RAB32-dependent host defence pathway in mice. Cryo-electron tomography revealed tether formation between Salmonella-containing vacuoles and host mitochondria upon Salmonella infection, which was significantly impaired in LRRK2-deficient cells. This positions LRRK2 centrally within a host defence mechanism, which may have favoured selection of a common familial Parkinson's disease mutant allele in the human population.

Biofilm formation, antimicrobial resistance genes, and genetic diversity of Salmonella enterica subspecies enterica serotype Enteritidis isolated from food and animal sources in Iran.

OBJECTIVES: Salmonella enterica serovar Entritidis is an important pathogen in foodborne diseases and causes gastroenteritis. Several studies have investigated the genetic diversity of the strains of this bacterium. However, our knowledge of the discriminatory power of the molecular methods is limited. METHODS: In total, 34 strains of S. enteritidis were isolated from food related to animals. Antibiotic resistance of the strains, antibiotic resistance genes, and biofilm formation capacity of the strains were evaluated. For the genetic analysis of the strains, PFGE was performed using AvrII restriction enzyme. RESULTS: Among the tested antibiotics, cefuroxime, nalidixic acid, and ciprofloxacin showed the highest resistance rates (79.4%, 47%, and 44.2%, respectively). Only three antibiotic-resistance genes were identified in these strains (blaTEM: 67.6%, tetA: 9%, and sul2: 3%). In total, 91% of the strains were biofilm producers. Clustering of strains using AvrII for 26 samples with the same XbaI PFGE profile showed that these strains were in one clone and had high homogeneity. CONCLUSIONS: In conclusion, it is better to use a combination of several typing methods for typing strains that are genetically very close so that the results are reliable.

Implementation of routine genomic surveillance provided insights into a locally acquired outbreak caused by a rare clade of Salmonella enterica serovar Enteritidis in Queensland, Australia.

Salmonellosis is a significant public health problem globally. In Australia, Salmonella enterica serovar Enteritidis is one of the main causes of salmonellosis. This study reports how the implementation of routine genetic surveillance of isolates from human S. Enteritidis cases enabled identification of the likely source of an outbreak that occurred in a remote town in Far North Queensland, Australia. This study included patient, food and water samples collected during an outbreak investigation. S. Enteritidis of the novel sequence type 5438 was isolated from all seven patient samples and one bore water sample but not any of the food samples. Both whole-genome single nucleotide polymorphism (SNP) and core-genome multilocus sequence typing analysis revealed that S. Enteritidis isolated from outbreak-related patient samples and the bore water isolates clustered together with fewer than five SNP differences and ten allelic differences. This genetic relatedness informed the outbreak response team around public health interventions and no further cases were identified post-treatment of the bore water. This disease cluster was identified through the routine sequencing of S. Enteritidis performed by the state public health laboratory in an actionable time frame. Additionally, genomic surveillance captured a case with unknown epidemiological links to the affected community, ruled out a simultaneous outbreak in an adjacent state as the source and provided evidence for the likely source preventing further transmission. Therefore, this report provides compelling support for the implementation of whole-genome sequencing based genotyping methods in public health microbiology laboratories for better outbreak detection and management.

Etiological Survey and Traceability Analysis of a Foodborne Disease Outbreak of Salmonella Senftenberg in Guizhou Province.

To conduct a study that examined the molecular epidemiology and pathogenesis of Salmonella Senftenberg isolates associated with an outbreak of foodborne disease in Guizhou Province and to provide a reference basis for the traceability of foodborne salmonellosis outbreaks and clinical diagnosis and treatment in the province. Fourteen strains of suspected Salmonella isolated from patient stool and food samples were used for pathogenic identification and serotyping by biochemical and mass spectrometry methods. Fourteen types of antibiotics were tested for drug sensitivity by the microbroth dilution method, and molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS). After the sequencing data were spliced by SPAdes, the gene protein sequences were compared with the Comprehensive Antibiotic Research Database and Virulence Factor Database, drug resistance and virulence genes were predicted, and whole genome multilocus sequence typing (wgMLST) was performed. The results were compared with those for Salmonella strains of the same serotype from the past 5 years in China detailed on the TraNet website. All 14 strains were identified as Salmonella Senftenberg (with the antigenic formula 1,3,19:g,s,t:-), and in the PFGE cluster tree, the strains were divided into two band types, with a similarity of 88.9%. The 14 strains were sensitive to the 14 antibiotics. WGS analysis showed that the 14 strains carried the same drug resistance and virulence genes and that all strains carried 3 aminoglycoside and lipopeptide drug resistance genes, including 114 virulence genes. The wgMLST results showed that the strains were distributed on the same small branch as those obtained from previous outbreaks of infection in Tianjin and Jilin. Salmonella Senftenberg, which caused the outbreak, carries a variety of virulence genes, which suggests that the strain is highly pathogenic. These pathogenic bacteria may be associated with the Salmonella strain in Tianjin, Jilin, and other places and have caused foodborne disease outbreaks as a result of imported contamination.

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

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