Multilocus sequence typing, clinical characteristic, and drug resistance of Salmonella typhimurium in 93 children / 中华实用儿科临床杂志

Objective:To investigate the multilocus sequence typing (MLST), clinical manifestations, and drug resistance of Salmonella typhimurium in children, thus providing references for the diagnosis, treatment, and prevention of pediatric Salmonella typhimurium infection.Methods:The clinical data of patients with Salmonella typhimurium serotypes confirmed by stool or blood culture between November 2017 and October 2020 were retrospectively collected from Fujian Maternity and Child Health Hospital.MSLT and drug susceptibility test were performed on Salmonella typhimurium isolated from the samples. Kruskal- Wallis test, Chi- square test, and Fisher′ s exact probability method were employed for data analyses. Results:Salmonella typhimurium was cultured from clinical samples of 96 children, of which, 93 samples were effective, including 92 stool samples and 1 blood sample from 53 boys(56.99%) and 40 girls(43.01%). The median age of disease onset occurred at 12.0 (8.5-22.0) months, with peak months of onset ranging from July to October.According to MLST classification, 93 children were divided into ST34 classification( n=58), ST19 classification( n=22) and other classification( n=13). Respiratory symptoms were significant different among MLST classification, and ST34 type Salmonella typhimurium enteritis was more commonly accompanied by respiratory symptoms ( χ2=17.657, P<0.001; Cramer V=0.421, P<0.001). There were significant differences in the drug sensitivity to Ampicillin ( χ2=8.774, P=0.033), Piperacillin tazobactam ( χ2=6.713, P=0.022), Ciprofloxacin ( χ2=20.780, P<0.001), Sulfamethoxazole ( χ2=15.364, P=0.001), Ampicillin sulbactam ( χ2=17.626, P=0.001) and Levofloxacin ( χ2=25.648, P<0.001) among 3 groups.No significant difference was found in the sensitivity to Ceftriaxone ( χ2=1.027, P=0.621), Ceftazidime ( χ2=7.637, P=0.059), Cefepime ( χ2=6.099, P=0.116) and Cefoperazone/Sulbactam ( χ2=2.405, P=0.649). All MLST types were sensitive to Imipenem and Meropenem. Conclusions:Salmonella typhimurium infection mainly affects infants, with the peak months of onset ranging from July to October.MLST34 is the main serotype, accompanied respiratory symptoms.Antibiotics should be selected according to drug sensitivity, and third-generation cephalosporins can be selected empirically without drug susceptibility test results.

Use of organic acids to reduce Salmonella Typhimurium excretion in swine / Uso de ácidos orgânicos para reduzir a excreção de Salmonella Typhimurium em suínos

The use of antimicrobials as growth promoters and disease prevention is being constantly reduced in several animal production systems, including in the swine industry. Therefore, this study aimed to evaluate the effectiveness of using acidifiers to control Salmonella Typhimurium in 65-day-old pigs by detecting the pathogen in organs at euthanasia. For this, 24 piglets were divided into two experimental groups consisting of 12 piglets each. An untreated control group (G1) and a treatment group (G2) received a liquid organic acidifier in the drinking water for 10 days (D-5 to D5). Five days after the start of treatment (D0), all piglets were challenged with 106 CFU of Salmonella Typhimurium and assessed for 12 days (D12). Every three days (D3, D6, D9, and D12), three animals from each experimental group were euthanized and then submitted for necropsy. Samples from the intestines (ileum, cecum, mesenteric lymph nodes, and ileocolic lymph nodes), liver, spleen, and lungs were collected to isolate Salmonella. The results show that, numerically, Salmonella isolation in the organs of G2 was lower than in G1 and that the number of positive cecum samples in G1 (66.7%; 8/12) was statistically different from the number of positive models in G2 (16.7%; 2/12), with a reduction of 28.6% of the total cecum positive samples in the treated group compared to the control. Therefore, it was observed that the liquid organic acidifier product could reduce the colonization of organs by Salmonella Typhimurium. (AU)

Use of organic acids to reduce Salmonella Typhimurium excretion in swine / Uso de ácidos orgânicos para reduzir a excreção de Salmonella Typhimurium em suínos

The use of antimicrobials as growth promoters and disease prevention is being constantly reduced in several animal production systems, including in the swine industry. Therefore, this study aimed to evaluate the effectiveness of using acidifiers to control Salmonella Typhimurium in 65-day-old pigs by detecting the pathogen in organs at euthanasia. For this, 24 piglets were divided into two experimental groups consisting of 12 piglets each. An untreated control group (G1) and a treatment group (G2) received a liquid organic acidifier in the drinking water for 10 days (D-5 to D5). Five days after the start of treatment (D0), all piglets were challenged with 106 CFU of Salmonella Typhimurium and assessed for 12 days (D12). Every three days (D3, D6, D9, and D12), three animals from each experimental group were euthanized and then submitted for necropsy. Samples from the intestines (ileum, cecum, mesenteric lymph nodes, and ileocolic lymph nodes), liver, spleen, and lungs were collected to isolate Salmonella. The results show that, numerically, Salmonellaisolation in the organs of G2 was lower than in G1 and that the number of positive cecum samples in G1 (66.7%; 8/12) was statistically different from the number of positive models in G2 (16.7%; 2/12), with a reduction of 28.6% of the total cecum positive samples in the treated group compared to the control. Therefore, it was observed that the liquid organic acidifier product could reduce the colonization of organs by Salmonella Typhimurium.(AU)

O uso de antimicrobianos como promotores de crescimento e prevenção de doenças vem sendo constantemente reduzido em diversos sistemas de produção animal, inclusive na suinocultura. Portanto, o objetivo do presente estudo foi avaliar a eficácia do uso de acidificantes no controle de Salmonella Typhimurium em suínos de 65 dias de idade, detectando o patógeno em órgãos após a eutanásia. Para isso, 24 leitões foram divididos em dois grupos experimentais constituídos por 12 leitões cada. Um grupo controle não tratado (G1) e um grupo de tratamento (G2) que recebeu um acidificante orgânico líquido na água de beber por 10 dias (D-5 a D5). Cinco dias após o início do tratamento (D0), todos os animais foram inoculados oralmente com 106 UFC de Salmonella Typhimurium e avaliados por 12 dias (D12). A cada três dias (D3, D6, D9 e D12), três leitões de cada grupo experimental foram eutanasiados e posteriormente submetidos à necropsia. Amostras de intestino (íleo, ceco, linfonodos mesentéricos e linfonodos ileocólicos), fígado, baço e pulmões foram coletadas para o isolamento de Salmonella. Os resultados mostram que, numericamente, o isolamento de Salmonella nos órgãos do G2 foi inferior ao G1, e que o número de amostras positivas de ceco no G1 (66,7%; 8/12) foi estatisticamente diferente do número de amostras positivas no G2 (16,7%; 2/12), com redução de 28,6% do total de amostras positivas de ceco no grupo tratado em relação ao controle. Portanto, observou-se que o ácido orgânico líquido foi capaz de reduzir a colonização de órgãos por Salmonella Typhimurium.(AU)

Mutagenic Evaluation of Fluoxetine and Fluoxetine-Galactomannan Complex Through the Analysis of Chromosomal Aberrations in Human Peripheral Leukocytes and Salmonella typhimurium/Microssome Assay / Avaliação Mutagênica de Fluoxetina e Complexo Fluoxetina-Galactomanana Através da Análise de Aberrações Cromossômicas em Leucócitos Periféricos Humanos e em Salmonella typhimurium / Ensaio Microssômico

Objectives: The purpose of this study was to evaluate the mutagenic potential of fluoxetine and fluoxetine-galactomannan. Methods: Chromosomal aberration test and Salmonella typhimurium/microsome mutagenicity assay. Results: The results showed that fluoxetine (250 µg/mL) can cause chromosomal breaks of treated leukocytes and increase the frequency of reversion of the tester strains of S. typhimurium / microsome assay only at the highest concentration (5 mg/mL), while fluoxetine encapsulated in galactomannan did not cause these changes (leukocytes and S. typhimuriums strains). Conclusion: In summary, fluoxetine showed a mutagenic effect detectable only at high concentrations in both eukaryotic and prokaryotic models. Furthermore, the fluoxetine/galactomannan complex, in this first moment, prevented the mutagenicity attributed to fluoxetine, emphasizing that the present encapsulation process can be an alternative in preventing these effects in vitro.

Objetivos: avaliar o potencial mutagênico da fluoxetina e da fluoxetina-galactomanana. Métodos: Teste de aberração cromossômica e ensaio de mutagenicidade de Salmonella typhimurium /microssoma. Resultados: a fluoxetina (250 µg/mL) pode causar quebras cromossômicas de leucócitos tratados e aumentar a frequência de reversão das cepas testadoras de S. typhimurium /microssoma apenas na concentração mais alta (5 mg/mL), enquanto a fluoxetina encapsulada em galactomanano não causou essas alterações (leucócitos e cepas de S. typhimurium). Conclusão: a fluoxetina mostrou um efeito mutagênico detectável apenas em altas concentrações em modelos eucarióticos e procarióticos. Além disso, o complexo fluoxetina/galactomanan, neste primeiro momento, evitou a mutagenicidade atribuída à fluoxetina, ressaltando que o presente processo de encapsulamento pode ser uma alternativa na prevenção desses efeitos in vitro.

In Silico Modeling and Docking Analysis of CTX-M-5, Cefotaxime-Hydrolyzing ?-Lactamase from Human-Associated Salmonella Typhimurium

Objective: To computationally model the CTX-M-5 ?-lactamase and establish its structure, which is exclusively present in human-associated Salmonella. Methods: The CTX-M-5 aminoacid sequence (Uniprot ID:O65975) of Salmonella enterica subsp. enterica serovar typhimurium was retrieved from UniProt database and subjected to homology modeling using MODELLER 9v7. The homology models were duly validated using RAMPAGE tool by generating Ramachandran plots, ERRAT graphs, and ProSA score. DoGSiteScorer server and ConSurf server were used to detect the cavities, pockets, and clefts to identify conserved amino acid sites in the predicted model. Subsequently, the modeled structure was docked using CLC Drug Discovery Workbench against proven drugs and known inhibitors. Results: Obtained high-quality homology model with 91.7% of the residues in favorable regions in Ramachandran plot and qualified in other quality parameters. Docking studies resulted in a higher dock score for PNK (D-benzylpenicilloic acid) molecule when compared to other reported inhibitors. Conclusion: This in silico study suggests that the compound PNK could be an efficient ligand for CTX-M-5 ?-lactamase and serve as a potent inhibitor of CTX-M-5.

Capability of acidic electrolyzed water in the elimination of Salmonella Typhimurium and Escherichia coli in the chicken breast

Aims@#This study aimed to investigate the effect of acidic electrolyzed water (AEW) as pre-refrigeration and pre-freezing processing steps for chicken meat in regard to the behavior of S. Typhimurium and E. coli during storage.@*Methodology and results@#AEW (free available chlorine 30 ppm and pH 2.7) was tested against S. Typhimurium and E. coli in growth media (brain heart infusion broth) and by exposing inoculated chicken fillets. The in vitro study appointed 10 minutes as the straightening exposure time of fresh prepared AEW for S. Typhimurium and E. coli. The reduction effect of AEW was significant (p<0.05) for both S. Typhimurium and E. coli along the 8 days of refrigerated storage with a maximum reduction after 24 h of post-treatment reaching 23.3% (1.4 log CFU/g) and 32.43% (2.15 log CFU/g) for S. Typhimurium and E. coli, respectively. AEW resulted in a significant reduction (p<0.05) as a pre-freezing application for both microorganisms, where the maximum reductions of 20% (1.2 log CFU/g) and 31.84% (2.14 log CFU/g) for S. Typhimurium and E. coli, respectively, were reported at zero time (just after dipping). In exposed samples to AEW, S. Typhimurium could not be detected by the 6th week of frozen storage while E. coli continued detectable until till 10th week but with a reduced population of 30% compared to control.@*Conclusion, significance and impact of study@#The findings of the present study suggest the application of AEW as a pre-refrigeration and pre-freezing treatment for chicken products. AEW application significantly improved the safety of chicken products.

Antimicrobial resistance and molecular typing of Salmonella typhimurium in Wuxi city, 2011-2018 / 中华检验医学杂志

Objective:Analysis of the antibiotic resistance profiles and molecular typing of Salmonella typhimurium ( S. typhimurium) isolated in Wuxi city from 2011 to 2018. Methods:A total of 109 S. typhimurium isolates were detected from different types monitoring samples in Wuxi city from 2011 to 2018. Microbroth dilution method was used to test antimicrobial susceptibility of S. typhimurium for 17 antimicrobial agents. Pulsed field gel electrophoresis (PFGE) was used to conduct molecular typing. Statistical analysis was carried out using Chi-square test. Results:Tetracycline-resistance and ampicillin-resistance were most frequency in 109 S. typhimurium isolates, 69.72% (76/109) and 68.81% (75/109), respectively. For compound sulfamethoxazole, chloramphenicol, ciprofloxacin, ampicillin/sulbactam, azithromycin, nitrofurantoin, cefotaxime and aztreonam, the resistance rates were 23.85%(26/109), 22.02%(24/109), 11.93%(13/109), 4.59%(5/109), 3.67%(4/109), 3.67%(4/109), 0.92%(1/109), 0.92%(1/109), respectively. All isolates were susceptible to amikacin, ertapenem, imipenem, meropenem, ceftazidime and ceftazidime-avibactam. Azithromycin-resistance isolates were decreasing year by year gradually. The aztreonam and cefotaxime-resistant isolates were found since 2018, while chloramphenicol and compound sulfa-resistant isolates showed upward trend simultaneously. Conclusions:S. typhimurium in Wuxi city exhibited highly resistant to tetracycline and ampicillin. The significant variability existed between genotype and phenotype of S. typhimurium.

Antimicrobial activity and chemical characterization of Laurel essential oil / Atividade antimicrobiana e caracterização química do óleo essencial de Louro / Actividad antimicrobiana y caracterización química del aceite esencial de Laurel

The factors that arouse interest in the study of essential oils as biocidal agents are numerous, such as the fact that they have antibacterial, antifungal, insecticidal, antioxidant, anti-inflammatory and larvicidal properties. The objective of this work was to evaluate the antimicrobial activity, in vitro, of the laurel (Laurus nobilis L) essential oil on the growth of pathogenic bacteria Salmonella enterica serovar Typhimurium ATCC 14028 and Staphylococcus aureus ATCC 25923, at different exposure times, as well as to perform the chemical characterization. Twenty compounds were identified and quantified, representing 96.57% of the total composition. The class of oxygenated monoterpenes represented the majority class of the essential oil, with 1,8-cineol (33.8%) as the substance found in greater quantity, followed by linalool (17.79%). The third constituent in greater quantity was sabinene (12.23%), belonging to the group of monoterpene hydrocarbons. Terpinyl acetate (9.41%) was also considered to be quantitatively representative. Laurel essential oil showed bacteriostatic activity against S. Typhimurium ATCC 14028 and S. aureus ATCC 25923.(AU)

Os fatores que despertam interesse no estudo dos óleos essenciais como agentes biocidas são inúmeros, como o fato de possuírem propriedades antibacteriana, antifúngica, inseticida, antioxidante, antiinflamatória e larvicida. O objetivo deste trabalho foi avaliar a atividade antimicrobiana, in vitro, do óleo essencial de louro (Laurus nobilis L) sobre o crescimento das bactérias patogênicas Salmonella enterica sorovar Typhimurium ATCC 14028 e Staphylococcus aureus ATCC 25923, em diferentes tempos de exposição, assim como realizar a caracterização química do óleo. Vinte compostos foram identificados e quantificados, representando 96,57% da composição total. A classe dos monoterpenos oxigenados representou a classe majoritária do óleo essencial, sendo o 1,8-cineol (33,8%) a substância encontrada em maior quantidade, seguido do linalol (17,79%). O terceiro constituinte em maior quantidade foi o sabineno (12,23%), pertencente ao grupo dos hidrocarbonetos monoterpênicos. O acetato de terpinila (9,41%) também foi considerado quantitativamente representativo. O óleo essencial de louro apresentou atividade bacteriostática contra S. Typhimurium ATCC 14028 e S. aureus ATCC 25923.(AU)

Los factores que despiertan interés en el estudio de los aceites esenciales como agentes biocidas son innumerables, como el hecho de que tienen propiedades antibacterianas, anti fúngicas, insecticidas, antioxidantes, antiinflamatorias y larvicidas. El objetivo de este trabajo ha sido evaluar la actividad antimicrobiana, in vitro, del aceite esencial de laurel (Laurus nobilis L) sobre el crecimiento de bacterias patógenas Salmonella enterica serovar Typhimurium ATCC 14028 y Staphylococcus aureus ATCC 25923, en diferentes momentos de exposición, así como realizar la caracterización química del aceite. Se identificaron y cuantificaron veinte compuestos, que representan el 96,57% de la composición total. La clase de mono terpenos oxigenados representó la clase principal de aceite esencial, siendo el 1,8-cineol (33,8%) la sustancia que se encuentra en mayor cantidad, seguida del linalol (17,79%). El tercer constituyente en mayor cantidad fue el sabineno (12,23%), perteneciente al grupo de los hidrocarburos monoterpénicos. El acetato de terpinilo (9,41%) también se consideró cuantitativamente representativo. El aceite esencial de laurel mostró actividad bacteriostática contra S. Typhimurium ATCC 14028 y S. aureus ATCC 25923.(AU)

The influence of dehydroepiandrosterone on effector functions of neutrophils

Dehydroepiandrosterone (DHEA) is a steroid hormone secreted by the adrenal glands, gonads and brain. It is a precursor to sex hormones and also is known to have immune modulatory activity. However, little is known about the relationship between DHEA and neutrophils and thus our study evaluates the influence of DHEA in the effector functions of neutrophils. Human neutrophils were treated in vitro with DHEA and further infected with Salmonella enterica serovar Typhimurium. The treatment of neutrophils with 0.01 µM of DHEA increased the phagocytosis of Salmonella independent of TLR4 as the treatment did not modulate the TLR4 expression. Additionally, DHEA caused a decrease in ROS (reactive oxygen species) production and did not influence the formation of the neutrophil extracellular trap (NET). Steroid treated neutrophils, infected or stimulated with LPS (lipopolysaccharide), showed reduced production of IL-8, compared to untreated cells. Also, the protein levels of p-NFκB were decreased in neutrophils treated with DHEA, and this reduction could explain the reduced levels of IL-8. These results led us to conclude that the steroid hormone DHEA has important modulatory functions in neutrophils

Caracterización molecular y fenotípica de aislamientos clínicos de Salmonella Typhimurium variante monofásica (1, 4, [5],12: i: -) recuperados en Colombia / Molecular and phenotypic characterization of Salmonella Typhimurium monophasic variant (1, 4, [5],12: i: -) from Colombian clinical isolates

Resumen: Introducción. La variante monofásica (1,4,[5],12:i:-) de Salmonella Typhimurium ocupa los primeros lugares en los programas de vigilancia de Salmonella a nivel mundial. En Colombia, Salmonella enterica variante monofásica alcanza el cuarto lugar en cuanto a los aislamientos clínicos recuperados por medio de la vigilancia por laboratorio del Grupo de Microbiología del Instituto Nacional de Salud, pero se desconoce si dichos aislamientos están relacionados con la variante monofásica de Typhimurium que circula a nivel global, y con sus características genéticas y fenotípicas. Objetivo. Caracterizar los aislamientos de Salmonella monofásica recuperados en Colombia entre el 2015 y el 2018 por el Grupo de Microbiología del Instituto Nacional de Salud. Materiales y métodos. Se analizaron 286 aislamientos clínicos de Salmonella enterica variante monofásica mediante PCR o secuenciación del genoma completo (Whole Genome Sequencing, WGS) para confirmar si correspondían a Salmonella Typhimurium variante monofásica, en tanto que, en 54 aislamientos, se determinó la estructura genética del operón que codifica la segunda fase flagelar y, en 23, se evaluó la motilidad, el crecimiento y la expresión de las proteínas de membrana externa. Resultados. El 61 % (n=174) de los aislamientos de Salmonella monofásica correspondió a Salmonella Typhimurium serovar monofásico. El 64,8 % (n=35/54) se relacionó con el clon europeo-español y, el 13 % (n=7/54), con el estadounidense. En dos aislamientos de orina se encontró una diferencia significativa en la motilidad y el crecimiento, así como ausencia de la porina OmpD en medio mínimo M9. Conclusiones. En el periodo de estudio, circuló en Colombia la variante monofásica de Salmonella Typhimurium relacionada con el clon europeo-español, y se registró ausencia total del operón fljAB. Los resultados evidenciaron cambios fenotípicos en los aislamientos provenientes de muestras de orina que sugieren adaptación en procesos invasivos.

Abstract: Introduction. The Salmonella Typhimurium monophasic variant (1,4,[5],12:i:-) is currently the most commonly detected variant in Salmonella surveillance programs worldwide. In Colombia, the Salmonella enterica monophasic variant is the fourth most common clinical isolate recovered through the laboratory surveillance of the Grupo de Microbiología from the Instituto Nacional de Salud; however, it is unknown whether these isolates are closely related to the monophasic Typhimurium variant, which circulates globally, and their genetic and phenotypic characteristics have not been reported. Objective. To characterize monophasic Salmonella enterica isolates identified in Colombia from 2015 to 2018 by the Instituto Nacional de Salud. Materials and methods. Two hundred eighty-six clinical isolates of the monophasic Salmonella enterica variant were analyzed by PCR or whole-genome sequencing to confirm whether they corresponded to the Salmonella Typhimurium monophasic variant while the genetic structure of the operon encoding the second flagellar phase was determined in 54 isolates. Motility, growth, and expression of the outer membrane proteins were evaluated in 23 isolates. Results. During the study period in Colombia, 61% (n=174) of Salmonella monophasic isolates belonged to Salmonella Typhimurium serovar monophasic (1,4,[5],12:i-). Of these, 64.8% (n=35/54) were related to the European/Spanish clone and 13% (n=7/54) to the U.S. clone. Two isolates recovered from urine samples showed differences in motility, growth, and the absence of the OmpD porin in M9 minimal medium. Conclusions. Most of the monophasic Salmonella Typhimurium variants that have circulated in Colombia since 2015 lacked the second phase of operon fljAB, which is related to the European/Spanish clone. The results evidenced phenotypic changes in urine samples suggesting bacterial adaptation in the case of these invasive samples.

Emerging Threat of Salmonella typhimurium in Extraintestinal Infections: Cases from a Tertiary Care Hospital in North India

Purpose:Infections due to invasive non-typhoid salmonella can be dangerous and fatal. The mode of infection and the severity varies from the typhoidal fevers. It is important to find the association between clinical features and the infecting serovar to understand the pathophysiology and course of treatment Methods:In the present study, extra-intestinal specimens (blood, cerebrospinal fluid and pus) from three patients suffering from septicaemia, meningitis and osteomyelitis were received. Micro-biological and biochemical test for species identification and antibiotic susceptibility was done as per standard protocol.Further, PCR based amplification and sequencing of a portion of the flagellin gene (FliC) was done to confirm the serovar.Results: Salmonellaentericawas identified from all the threeby microbiological and biochemical examination.The sequence of the Flic gene confirmed the serovar to be S.typhimurium. All the patients were treated successfully for the infectionby appropriate antibiotic therapy. Conclusion:The study highlights that serovarTyphimurium is common in invasive non-typhoidal salmonellosis and its pathophysiology and virulence factors expression should be understood in various organ types for better treatment options and outcomes

Expressão e atividade de sistemas de dois componentes (TCS) de Salmonella enterica sorovar Typhimurium no contexto do metaboloma intestinal humano / Expression and activity of two-component systems of Salmonella enterica sorovar Typhimurium in the context of the human intestinal metabolome

A interação entre membros do microbioma intestinal humano, células hospedeiras e patógenos invasores pode ocorrer de diversas formas, sendo uma delas através de pequenas moléculas chamadas metabólitos. A percepção e resposta efetiva de um microrganismo às diferentes condições encontradas em seu ambiente, incluindo metabólitos produzidos por outros microrganismos, são fatores importantes para sua adaptação, sobrevivência e disseminação. Os sistemas de dois componentes (TCS) permitem a percepção e resposta a mudanças ambientais, regulando a expressão de genes específicos. Nosso grupo mostrou anteriormente que um extrato orgânico de fezes humanas (EF), bem como o ácido 3,4-dimetilbenzoico (3,4-DMB), encontrado no EF, inibe a capacidade de Salmonella enterica sorovar Typhimurium de invadir células hospedeiras. O presente trabalho propôs investigar o impacto do microbioma intestinal humano, bem como de pequenas moléculas produzidas por Clostridium citroniae (membro deste microbioma) na expressão e atividade dos genes de TCS de Salmonella. Os metabólitos de EF e de culturas puras de C. citroniae foram extraídos com acetato de etila e adicionados a meio de cultura. O pH do meio foi ajustado (~ 7,4) e a solução foi esterilizada por filtragem. Salmonella foi cultivada na presença ou ausência do EF e do extrato de C. citroniae, bem como do ácido 3,4-DMB, em condições aeróbias e anaeróbias, até alcançar o meio da fase logarítmica de crescimento. O RNA foi extraído para a realização de PCR em Tempo Real utilizando iniciadores direcionados a quase todos os TCS de Salmonella. Nossos resultados mostraram que vários genes de TCS envolvidos na virulência de Salmonella (SsrAB, EnvZ-OmpR, QseCB, PhoQP, TorSR, TtrRS) foram regulados diferencialmente por esses metabólitos, tanto em condições aeróbias quanto anaeróbias. EnvZ-OmpR, PhoPQ e SsrAB estão diretamente envolvidos na regulação das Ilhas de Patogenicidade 1 e 2 de Salmonella. QseCB é crucial para a detecção de quorum em Salmonella, de hormônios hospedeiros e para a regulação da motilidade (swimming). Vários outros TCS também foram regulados, incluindo TorSR e TtrRS, envolvidos na regulação da respiração anaeróbica de N-óxido de trimetilamina (TMAO) e tetrationato, respectivamente. Esses compostos são importantes para a sobrevivência de Salmonella no ambiente anaeróbico do intestino humano. Nossos resultados de avaliação de expressão gênica global de Salmonella cultivada na presença de ácido 3,4-DMB (aerobiose e anaerobiose) bem como na presença do EF em anaerobiose, mostraram que genes condificados em SPI-1 e SPI-2, SPI-4 e alguns genes do TCS foram reprimidos, enquanto genes marR, marB e marA foram ativadas nessas condições. Adicionalmente, comparamos nossos resultados de RNAseq, de Salmonella cultivada na presença do ácido 3,4-DMB em aerobiose, com resultados disponíveis da base de dados Salmonella Compendium. Ainda, a capacidade de Salmonella de adentrar e sobreviver dentro de células fagocíticas (macrófagos RAW 264.7) parece ser afetada pelas três condições testadas neste trabalho. Nossos resultados mostram que importantes vias de sinalização da virulência de Salmonella podem ser moduladas pelos metabólitos presentes no microbioma intestinal humano e abrem caminhos para novas pesquisas sobre a sinalização intercelular microbioma-patógeno no ambiente intestinal.

The interaction between members of the human gut microbiome, host cells and invading pathogens often occurs through small molecules, also called metabolites. The perception and effective response of a microorganism to the different conditions found in its environment, including metabolites produced by other microbes, is important for its adaptation, survival and dissemination. Two-component systems (TCS) allow the perception and response to environmental changes by regulating the expression of specific genes. Our group previously showed that organic extracts of human feces (EF) as well as the specific metabolite 3,4-dimethylbenzoic acid (3,4-DMB) found within the EF, inhibit the ability of Salmonella enterica sorovar Typhimurium to invade host cells. In the present work, we investigated the impact of the human gut microbiome as well as small molecules produced by Clostridium citroniae (a member of this microbiome) on the expression and activity of Salmonella TCS genes. Metabolites (from feces or C. citroniae cultures) were extracted using ethyl acetate and added to culture medium. The pH of the medium was adjusted (~7.4), and the solution was filter sterilized. Salmonella was grown in the presence or absence of the organic extracts as well as 3,4-DMB acid under aerobic and anaerobic conditions until it reached mid-log growth. RNA was then extracted for Real-time PCR using primers targeting almost all Salmonella TCS. Our results showed that several TCS involved in Salmonella virulence (SsrAB, EnvZ-OmpR, QseCB, PhoQP, TorSR, TtrRS) were differentially regulated by these metabolites both in aerobic and anaerobic conditions. EnvZ-OmpR, PhoPQ, and SsrAB are directly involved in the regulation of Salmonella Pathogenicity Islands 1 and 2. QseCB is crucial for Salmonella =quorum sensing, sensing of host hormones and regulation of swimming motility. Several other TCS were also regulated, including TorSR and TtrRS, which are involved in the anaerobic respiration of trimethylamine N-oxide (TMAO) and tetrathionate, respectively. These compounds are important for Salmonella survival in the anaerobic environment of the human gut. Our results of the evaluation of global Salmonella gene expression grown in the presence of 3,4-DMB acid (aerobiosis and anaerobiosis) as well as in the presence of EF in anaerobiosis, showed that genes encoded in SPI-1 and SPI-2, SPI-4 and some TCS genes have been repressed, while multiple drug resistance genes, as well marR, marB and marA genes have been activated under these conditions. Besides, we compared our results of RNAseq, Salmonella was grown in the presence of 3,4-DMB acid in aerobiosis, with results available from the Salmonella Compendium database. Also, Salmonella's ability to enter and survive within phagocytic cells (macrophages RAW 264.7) appears to be affected by the three conditions tested in this work. Our results show that important Salmonella virulence signalling pathways can be modulated by the metabolites present in the human intestinal microbiome and open the way for further research on the microbiome-pathogen intercellular signalling in the intestinal environment.

Construction and characterization of △sseK2 mutant strain of Salmonella typhimurium SL1344 / 中华微生物学和免疫学杂志

Objective To analyze the biological characteristics of a mutant strain of Salmonella ty-phimurium SL1344 with sseK2-deletion (SL1344△sseK2) in order to provide reference for further study of safe and effective live vaccines. Methods The mutant strain SL1344△sseK2 with a deletion of 1047 bp in sseK2 gene was constructed through a two-step allelic exchange using recombinant suicide plasmid. Its com-plemented strain, SL1344C△sseK2, was also constructed. Biological and immunological characteristics of the mutant strain were detected. Results PCR, double-enzyme digestion and sequencing analysis showed that the mutant strain SL1344△sseK2 and the complemented strain SL1344C△sseK2 were successfully con-structed. The serotype of the mutant strain was 1,4,[5],12:i:1,2, identical to the parent strain SL1344. In addition, the mutant strain showed no significant change in biochemical characteristics or growth rate and was genetically stable in vitro. Compared with the parent strain SL1344, the virulence of SL1344△sseK2 was attenuated in BALB/ c mice. The median lethal dose of SL1344△sseK2 for 6-week-old BALB/ c mice was 3. 44×108 colony-forming units (CFU), which was 1620 times lower than that of SL1344. Oral immuniza-tion with SL1344△sseK2 protected 62. 5％ of the mice against challenge with wild Salmonella typhimurium strains on 17 d after vaccination. The levels of serum IgG antibody peaked on 14 d after immunization. No significant difference in biological characteristics was observed between the complemented and the parent strains, indicating that the mutant strain was basically complemented to the wild-type strain.Conclusions The mutant strain SL1344△sseK2 was constructed successfully and genetically stable with sig-nificantly attenuated virulence and good immunogenicity. This study suggested that sseK2 gene played an im-portant role in regulating the virulence of SL1344, which might provide reference for further study of its func-tion and for assessing its potential as a candidate live attenuated vaccine.

Molecular typing, virulence genes and drug resistance of Salmonella typhimurium in Longyan city / 中华微生物学和免疫学杂志

Objective@#To analyze the molecular typing characteristics by pulsed field gel electrophoresis (PFGE), drug resistance and virulence genes of Salmonella typhimurium in Longyan city in order to provide reference for the prevention and control.@*Methods@#A total of 79 Salmonella typhimurium strains were isolated from sporadic cases of diarrhea and food poisoning and raw poultry meat samples during 2010 to 2017. PFGE was performed to measure the minimum inhibitory concentrations (MIC) of 15 commonly used drugs against them. PCR was used to detect nine virulence genes (sopB, invA, sifA, sscA, sseE, spvB, pefA, spvR, spvC) in 55 strains.@*Results@#The 79 Salmonella typhimurium strains belonged to 61 PFGE types. There were 10, three and four strains of P1, P3 and P21 types, respectively. Seven P1 type strains were isolate from one food poisoning event. According to the 85% classification standard, 79 Salmonella typhimurium strains could be divided into five predominant gene clusters (G1-G5). Drug susceptibility test showed that the 79 strains had the highest resistance rate to ampicillin (88.61%), followed by that to tetracycline (87.34%) and streptomycin (73.41%). Multidrug resistant bacteria resistant to three or more antibacterial drugs accounted for 84.81% (67/79). All of the 55 strains carried invA, sopB, sseE and sscA genes. The other five genes, sifA, spvC, spvB, spvR and pefA, were detected in 54, 31. 10, 11 and 12 strains, respectively. There were 76.4% (42/55) of the strains carrying five or six virulence genes and all were positive for invA, sopB, sseE, sscA and sifA, and negative for spvB, spvR and pefA. The strains carrying all of the nine virulence genes accounted for 18.2% (10/55).@*Conclusions@#Salmonella typhimurium strains isolated in Longyan city had a diverse PFGE type. P1, P3 and P21 types were the three predominant PFGE types. In the food poisoning event, PFGE molecular typing could quickly alert the outbreak and traceability of Salmonella typhimurium. Attention should be paid to the multidrug resistance in Salmonella typhimurium. Monitoring of multidrug-resistant strains and supervision on antibacterial drug usage should be strengthened. Salmonella typhimurium had high virulence as it carried many virulence genes.

Anti-inflammatory actions and Salmonella typhimurium-bacteraemia clearance by methanol extract of Curcuma longa Linn. (Turmeric)

Aims@#Rhizome of turmeric is known to possess therapeutic activities and has been used in medical practice as an anti-diabetic, hypolipidemic, hepatoprotective, anti-diarrheal, and anti-asthma agent. This study was designed to investigate the anti-inflammatory and antimicrobial activities of Curcuma longa. @*Methodology and results@#Rhizomes of Curcuma longa (Turmeric) purchased from markets in Ado-Ekiti, Nigeria, were analysed for anti-inflammatory and anti-microbial properties, as well as phytochemical constituents. The in vitro anti-inflammatory activities of the C. longa methanol extract (CLME) were evaluated by albumin denaturation, proteinase inhibitory activity, membrane stabilization, and anti-lipoxygenase activity, at different concentrations using Aspirin, Diclofenac sodium and Indomethacin as standard drugs. The in vitro antimicrobial activities of CLME were carried out on five pathogenic microbes namely Escherichia coli ATCC 29929, Staphylococcus aureus ATCC 29293, Salmonella typhimurium ATCC 14028, Klebsiella pneumoniae ATCC 4252 and Candida albicans ATCC 10231, using both agar well diffusion and broth dilution techniques. A S. typhimurium infected rat model was used for in vivo antimicrobial studies. Phytochemical analyses showed that C. longa rhizomes contain high concentrations of alkaloids, flavonoid and saponins, with moderate levels of phenols, tannin and ferric reducing antioxidant power. CLME was found to be rich in alkaloids, tannins, phenols, steroids, saponins, terpenoids, flavonoids and reducing sugars. CLME showed potent anti-inflammatory activities, and the results compared favourably with the standard anti-inflammatory drugs used. C. longa methanol extract significantly inhibited albumin denaturation and proteinase activity, stabilized membrane of red blood cell from haemolysis in heat and hypotonic conditions, as well inhibited lipoxygenase activity; all of which are associated with inflammatory processes. CLME was found to possess high in vitro antimicrobial activities against the five microorganisms tested. Rats orally infected with S. typhimurium, demonstrated bacteraemia five days post infection, with a total clearance of bacteraemia within 3-5 days following oral administration of CLME. The infected rats treated with CLME equally showed significant improvement in some haematological indices compared to infected rats that were not treated with CLME. @*Conclusion, significance and impact of study@#The results also showed that methanol extract of C. longa rhizome effectively cured with S. typhimurium infected rats. The overall results suggest that Curcuma longa is a potential source of anti-inflammatory and antimicrobial agents.

Role of methylation in regulating PhoP activity in Salmonella enterica serovar Typhimurium / 上海交通大学学报（医学版）

Objective :To investigate the correlation between methylation and the activity of transcriptional regulator PhoP in Salmonella enterica serovar Typhimurium (S. Typhimurium), and screen for the methyltransferase of PhoP. Methods :The methylation level of PhoP in S. Typhimurium under different culture conditions was determined by Western blotting. The transcription levels of phoP and the genes regulated by phoP were examined to screen for the methyltransferase of PhoP after overexpressing methyltransferase candidates predicted by bioinformatics. And then methyltransferase assay was verified in vitro. The transcription levels of phoP and its methyltransferase were determined by real-time PCR in high concentration of Mg2+ or weak acid conditions. Results :As the concentration of Mg2+increased in the medium, the methylation level of PhoP increased, and its methylation level decreased after S. Typhimurium was stimulated by weak acid. In the screening of 9 methyltransferases predicted by bioinformatics, overexpression of STM14_0023 reduced the transcription level of phoP and its downstream genes and the protein level of PhoP in vivo, but knockout of STM14_0023 had no effect on the expression of phoP and its downstream genes. STM14_0023 was capable of methylating PhoP in vitro and could also increase the methylation level of PhoP after overexpression of STM14_0023 in vivo. Under the condition of high concentration of Mg2+ when the expression of phoP was inhibited, the transcriptional level of STM14_0023 was reduced. Conclusion :STM14_0023 is the methyltransferase of PhoP, and methylation inhibits PhoP activity.

Antisalmonellal and antioxidant potential of hydroethanolic extract of Canarium schweinfurthii Engl. (Burseraceae) in Salmonella enterica serovar Typhimurium-infected chicks

Objective: To evaluate the anti-infectious and antioxidant activities of hydroethanolic extract of Canarium schweinfurthii on broiler chickens infected by Salmonella enterica serovar Typhimurium, known to be threatening public health. Methods: Animals were divided into six groups of eight animals per group: the normal control group, negative control group, positive control group and three test groups receiving the plant extract at 5, 19 and 75 mg/kg bw, respecively. The evolution of the disease as well as the effectiveness of the treatment were monitored by stool culture from the second day post infection until the end of the treatment. In addition, the effects of treatment on growth performances and feed conversion efficiency of broilers were evaluated. For the assessment of antioxidant status, enzymatic and non-enzymatic biomarkers such as catalase, glutathione peroxidase, malondialdehyde and nitric oxide were evaluated in the serum and tissues of animals. Results: The infected chickens treated with oxytetracycline recovered on day 7 after treatment, while animals treated with 19 and 75 mg/kg of Canarium schweinfurthii extract recovered on day 9 and those with 5 mg/kg of the extract on day 10. Salmonella infection caused a decrease on catalase and glutathione peroxidase activities; the administration of various doses of Canarium schweinfurthii extract increased these enzymatic activities. Animals receiving the extract at 5 mg/kg showed a significant increase in catalase activity in serum, heart and lungs while all concentrations of the extract significantly increased glutathione peroxidase activity in the serum, liver and spleen. Concerning non-enzymatic biomarkers, Salmonella infection caused a significant increase of nitric oxide and malondialdehyde concentration in the liver and lungs. Treatment with 75 mg/kg of the extract significantly reduced nitric oxide concentration in the heart and lungs while each dose of the extract reduced and normalized the malondialdehyde level in the serum. Additionally, malondialdehyde production was significantly decreased in the liver, heart and lungs after administration of Canarium schweinfurthii extract at all doses. Conclusions: The hydroethanolic extract of Canarium schweinfurthii attenuates oxidative stress, and is effective in the treatment of avian salmonellosis.

Increasing prevalence and dissemination of invasive nontyphoidal Salmonella serotype Typhimurium with multidrug resistance in hospitalized patients from southern Brazil

ABSTRACT Introduction: Nontyphoidal Salmonella serotypes are the main cause of human food-borne infection, including several hospitalization cases in the developing countries. Aim: To detect the main serotypes and to characterize the antibiotic resistance of human non-enteric and enteric nontyphoidal Salmonella from clinical isolates in Brazil. Methods: Salmonella serotypes were identified by microbiological and molecular methods. Susceptibility testing to antibiotics was performed by agar disk diffusion. Real-time PCRs were carried out for the detection of the genus Salmonella as well as serotypes Typhimurium and Enteritidis. Results: A total of 307 nontyphoidal Salmonella were isolated from 289 different patients in a reference laboratory (LACEN-RS) from Southern Brazil in a six-year period (2010-2015). There were 45 isolates from emerging cases and 244 from sporadic cases in hospitalized patients. Non-enteric isolates were detected in 42.6% of the patients from sources such as urine, blood and other clinical fluids. Serological and PCR-specific tests demonstrated that Typhimurium (48.4%) and Enteritidis (18.3%) were the most frequent serotypes. Typhimurium isolates were generally resistant to three or more antibiotic classes, while Enteritidis isolates to one or two classes. Typhimurium was the most frequent serotype in all samples (48.4%), mainly among the hospitalized patients (55.6%), and presented the highest rates of multidrug resistance (59.3% of the isolates of this serotype). Further, the prevalence of this serotype increased along the years of the study in comparison to other nontyphoidal Salmonella serotypes. Conclusion: Greater public health attention should be given to prevent salmonellosis in the community and in hospital settings to reduce the rates of Typhimurium strains with multidrug resistance.

Comparison of three diagnostic methods for Salmonella enterica serovars detection in chicken rinse / Comparação de três métodos diagnósticos para detecção de Salmonella enterica em lavados de carcaça de frango

Salmonella detection is a key point in food safety testing, because of the frequent association of this pathogen with food poisoning in humans. The standard bacteriological tests currently used for Salmonella-detection are time-consuming; therefore, there is a need to develop alternative methods to accelerate the detection. In order to accelerate Salmonella diagnosis, we used the immunomagnetic separation assay associated with bacteriophage P22 for the rapid detection of the following Salmonella serovars in chicken rinses of drumsticks, artificially contaminated with 5, 10, and 100 CFU/25mL of bacteria: Salmonella enterica subsp. enterica serovar Heidelberg (S. Heidelberg), Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) and Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium). The efficiency of the technique, represented by the time required for detection of positive and negative samples, was compared with that of the standard diagnostic tests used for this pathogen, the bacteriological assay and the polymerase chain reaction (PCR)-based test. This study confirmed the ability of the bacteriophage-associated immunomagnetic separation assay to identify 99.6% of Salmonella-positive samples of the three serovars tested. In contrast, the bacteriological assay and PCR-based test detected 95.1% and 98.5% of the Salmonella-positive samples respectively.(AU)

A detecção de Salmonella é um ponto crucial para a segurança alimentar, devido a frequente associação deste patógeno com infecções alimentares em humanos. O método padrão para detecção de Salmonella é o bacteriológico, mas o tempo requerido para o processamento das amostras e o diagnóstico final é longo, por isso existe a necessidade de desenvolvimento de métodos alternativos que visem acelerar esta etapa. Para isto utilizamos a separação imunomagnética associada ao bacteriófago P22 como técnica de detecção rápida para os seguintes sorovares de Salmonella: Salmonella enterica subsp. enterica sorovar Heidelberg (S. Heidelberg), Salmonella enterica subsp. enterica sorovar Enteritidis (S. Enteritidis) e Salmonella enterica subsp. enterica sorovar Typhimurium (S. Typhimurium), os quais foram inoculados artificialmente em lavados de sobre-coxas de frango nas seguintes concentrações: 5, 10 e 100 UFC/25mL. A eficiência da técnica, representada pelo tempo requerido para detecção de amostras positivas ou negativas, foi comparado com os testes rotineiramente utilizados para detecção de Salmonella, o exame bacteriológico e a reação em cadeia da polimerase (PCR). Este estudo confirmou a capacidade do teste de separação imunomagnética associado a bacteriófago, o qual identificou 99,6% das amostras positivas para Salmonella, dos três sorovares testados. Já o bacteriológico e PCR identificaram respectivamente 95,1% e 98,5% das amostras positivas.(AU)

EFECTO DE LA COMBINACIÓN DE GLUCOSA OXIDASA/ GLUCOSA SOBRE EL CRECIMIENTO DE BACTERIAS DEL GÉNERO Salmonella AISLADAS DE AVES DE CORRAL / EFFECT OF THE GLUCOSE OXIDASE /GLUCOSE COMBINATION ON THE GROWTH OF THE GENUS Salmonella BACTERIA ISOLATED FROM POULTRY

RESUMEN En la industria avícola, las infecciones por Salmonella son un problema, debido a los efectos sobre la producción y los riesgos a la salud pública. En las aves infectadas puede ocurrir una diseminación sistémica de las bacterias, que afecta los órganos y favorece, tanto la transmisión entre las aves como la contaminación de los productos avícolas, tales como las carnes, durante el proceso de sacrificio de los animales. Se decidió evaluar el efecto que diferentes combinaciones de glucosa oxidasa (GOX) y glucosa tienen sobre el crecimiento de diversos serotipos de Salmonella aislados de aves en crecimiento y de engorde, y determinar su potencial como antibacteriano. Se tomaron muestras de corazón y de ciego de aves de 7 y 42 días de edad y se aislaron e identificaron S. typhimurium y S. enteritidis. Estas bacterias fueron cultivadas en medio Luria-Bertani (LB) en presencia de GOX (0,5; 1 y 2U/mL) y glucosa (0,5, 1 y 2%), en combinaciones bajo un diseño factorial 32. El crecimiento fue monitoreado por el cambio de absorbancia a 600 nm, durante 6 horas de incubación. Se observó una reducción significativa del crecimiento de ambos serotipos al utilizar 2U/mL de GOX y diferentes concentraciones de glucosa, lo cual, demostró la capacidad antibacteriana que posee el sistema GOX/glucosa sobre este género, por lo que se podría emplear como un aditivo para la conservación de carnes de aves y productos derivados, a fin de alargar su vida de anaquel y minimizar riesgos a la salud.

SUMMARY In the poultry industry, Salmonella infections are a problem due to the effects on production and risks to public health. In infected birds, a systemic spread of bacteria can occur, which affects the organs and favors both the transmission between birds and the contamination of poultry products, such as meat, during the animal´s slaughter process. It was evaluated the effect that different combinations of glucose oxidase (GOX) and glucose have on the growth of different Salmonella serotypes isolated from broilers, and thus determine their potential as antibacterial. Heart and caecal samples were taken from birds 7 and 42 days old. S. typhimurium and S. enteritidis were isolated and identified. These bacteria were cultured in Luria-Bertani medium (LB) in the presence of GOX (0.5, 1 and 2U/mL) and glucose (0.5, 1 and 2%) in combinations under a factorial design 32. Growth was monitored by absorbance change at 600nm for 6 hours of incubation. A significant reduction of the growth of both serotypes was observed when using 2 U/mL of GOX and different concentrations of glucose. This demonstrated the antibacterial capacity that the GOX/glucose system has on this genus, so it could be used as an additive for the preservation of poultry meats and derived products, in order to lengthen its storage time and minimize health risks.