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Background: Enteric fever is a major public health problem in developing countries like India. It affects all age groups but young children are at highest risk. Timely management with appropriate antimicrobial therapy can reduce both morbidity and mortality. In recent years, the emergence of antimicrobial resistance is a significant challenge. Therefore, this study was undertaken to study antibiotic sensitivity pattern of the Salmonella isolates. Aims and Objectives: The aim of this study was to estimate the prevalence of resistance to commonly used antibiotics in the Salmonella isolates. Materials and Methods: Children between 6 months and 14 years of age admitted for fever and whose blood culture was positive for Salmonella Typhi or Salmonella Paratyphi A, B, or C were included in the study over a period of 2 years (August 2018–July 2020). Results: There were 155 patients of enteric fever whose blood culture results were positive for S. Typhi/S. Paratyphi who were included in the study. Out of the 155 culture positive cases, S. Typhi was isolated in 135 (87.1%), S. Paratyphi A in 16 (10.3%) and S. Paratyphi B in 4 (2.6%) cases. All the 135 isolates of S. Typhi were sensitive to cephalosporins. High rate of sensitivity was noted for the first-line drugs – amoxicillin, ampicillin, and trimethoprim-sulfamethoxazole. All 16 isolates of S. Paratyphi A were sensitive to cephalosporins. All the isolates of S. Paratyphi B tested were sensitive to cephalosporins, azithromycin, nalidixic acid, and trimethoprim-sulfamethoxazole. Conclusion: At present, there is low prevalence of resistance to first-line drugs and third-generation cephalosporins and high resistance to fluoroquinolones, nalidixic acid, and azithromycin as noted in this region. Rational antibiotic selection should be based on sensitivity pattern to prevent emergence of resistant strains.
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La capacidad inmunoestimuladora de la mayoría de las vacunas es potenciada mediante la adsorción en adyuvantes que contienen aluminio. Variando las condiciones de adsorción (pH, tiempo de adsorción) cambia la cantidad de antígeno adsorbida y por lo tanto la capacidad de estimulación del sistema inmune. El Instituto Finlay de Vacunas investiga un nuevo candidato vacunal basado en vesículas de membrana externa de Salmonella Paratyphi A (VME-SPA). El objetivo de este trabajo fue determinar las condiciones de adsorción de las VME-SPA en dos adyuvantes de sales de aluminio (Al(OH)3 y AlPO4). Para ello, las VME-SPA fueron adsorbidas en ambos adyuvantes bajo diferentes condiciones de pH y tiempo. Mediante la construcción de una Isoterma de Langmuir se determinaron parámetros como la capacidad adsortiva (qm) y el coeficiente de adsorción (Kd). El lote de VME-SPA empleado estaba formado por poblaciones de nanoestructuras con un tamaño de partículas entre 60 y 100 nm. La adsorción de las VME-SPA en ambos adyuvantes, mostró valores ≥95 por ciento a pH neutro (6,5-7,0). Las VME-SPA en presencia de AlPO4 alcanzaron el estado de equilibrio en menor tiempo (99 por ciento a partir de 30 min) en comparación con Al(OH)3 (95 por ciento a partir de 3 h). Las isotermas evaluadas para ambos adyuvantes cumplieron con el modelo de Langmuir (R2≥0,99), con valores de qm y Kd diferentes entre los sistemas de adsorción. El estudio demostró que las VME-SPA se adsorbieron satisfactoriamente en ambos geles, proceso en el que están involucrados diferentes mecanismos de adsorción(AU)
The immunostimulation capacity of most vaccines is enhanced through antigen adsorption on aluminum adjuvants. The changes in adsorption conditions (pH, adsorption time), could change the amount of antigen adsorbed and therefore the ability to stimulate the immune system. The Finlay Institute of Vaccine researches a new vaccine candidate based on outer membrane vesicle from Salmonella Paratyphi A (OMV-SPA). The study aim was to determine adsorption condition of OMV-SPA with two aluminium adjuvants (Al(OH)3 and AlPO4). OMV-SPA was adsorbed in both adjuvants under differences conditions of pH and time. Parameters as adsorptive capacity (qm) and adsorption coefficient (Kd) were determined by construction of Langmuir Isotherm. The lot of OMV-SPA used is composed by population of nanostructure with a particle size between 60 and 100 nm. Adsorption of OMV-SPA in both adjuvants showed values ≥95 percent in neutral pH (6.5-7.0). OMV-SPA with AlPO4 got equilibrium state in less time (99 percent from 30 min) compared with Al(OH)3 (95 percent from 3 h). Isotherms from both adjuvants described Langmuir model (R2≥0.99), with qm and Kd values very different between adsorption systems. As conclusion, the study showed that OMV-SPA was adsorbed satisfactorily in both aluminium adjuvants, process in which are involved different adsorption mechanism(AU)
Subject(s)
Salmonella Vaccines/immunology , Aluminum Hydroxide , VaccinesABSTRACT
Background: Enteric fever or Typhoid fever is caused mostlyby Salmonella enterica serovar Typhi and Salmonella entericaserovar Paratyphi is an important public health challenge forIndia, especially with the spread of antimicrobial resistance.The situation is further complicated by increased incidence insome parts of the country of S. Paratyphi A as a cause ofenteric fever. This serovar is not prevented by currentlyavailable typhoid vaccines and represents an increasing threatto human health.Purpose: The present study was undertaken to analyse thetrend in prevalence of culture-positive typhoid fever during thelast three years and to determine antimicrobial susceptibilityprofile of Salmonella Typhi and Salmonella Paratyphi Aisolated from patients of enteric fever in this part of the country.Material: This retrospective study incorporates a three years,(January 2015-December2018) laboratory data comprising52isolates of Salmonella. Cultures were identified by standardmethods. Antimicrobial susceptibility was done againstchloramphenicol, amoxicillin, co-trimoxazole, ciprofloxacin,ceftriaxone, cefixime and azithromycin as per correspondingCLSI guidelines for each year.Results: Salmonella enterica serotype Typhi (S. Typhi) wasthe more frequent serotype isolated i.e., 73% with theremaining 27% being Salmonella enterica serotype Paratyphi A(S. Paratyphi A). There was emergence of S. Paratyphi Aserotype in 2017-18. Antimicrobial susceptibility forchloramphenicol, amoxicillin and co-trimoxazole, ciprofloxacinand ceftriaxone was found to be 97.2%, 88.5%, 90.5%, 66.8%,99.4% for S. Typhi and 100%, 90.1% and 92.8%, 71.5%, 100%for S. Paratyphi A.Conclusion: The present study confers Salmonella ParatyphiA as the rapidly emerging pathogen of enteric fever. Theantibiogram of Salmonella Typhi and Salmonella Paratyphi Ashowed decreased susceptibility to fluoroquinolones and anotable decrease in the multi drug resistant strains ofSalmonella isolates with re-emergence of susceptibility to firstline antibiotics.
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Salmonella Paratyphi A es un patógeno exclusivo de humanos, siendo la segunda causa más común de fiebre entérica en el sudeste asiático. Recientemente la incidencia en este continente ha aumentado, desplazando a Salmonella entérica serotipo Typhi como la primera causa de fiebre entérica. En la actualidad no existen vacunas licenciadas contra S. Paratyphi A. El Instituto Finlay de Vacunas se encuentra trabajando en la obtención de un candidato vacunal basado en vesículas de membrana externa (VME) contra S. Paratyphi A, por lo que se hizo necesario contar con una técnica para la evaluación de su inmunogenicidad. El objetivo de este trabajo fue la estandarización de un ELISA para la cuantificación de anticuerpos IgG contra VME de S. Paratyphi A. Para ello, se determinaron las mejores condiciones de este ensayo en cuanto a concentración óptima de recubrimiento y dilución de trabajo del conjugado. Además, se definió el intervalo y linealidad de la curva, la precisión intra e interensayo, la especificidad y el límite de detección. La curva de calibración se generó con un suero estándar interno y presentó un buen ajuste lineal con un R² =0.98. Los coeficientes de variación en los ensayos de precisión intra e interensayo estuvieron en los intervalos establecidos para cada uno (=10 por ciento, =20 por ciento respectivamente). Los resultados obtenidos avalan el empleo de este ELISA cuantitativo para la evaluación de la inmunogenicidad de formulaciones de VME de S. Paratyphi A en fases de investigación y desarrollo(AU)
Salmonella Paratyphi A, is an exclusive pathogen of humans, being the second most common cause of enteric fever in Southeast Asia. Recently the incidence of this disease in this continent has increased, displacing Salmonella enterica serotype Typhi as the first cause of enteric fever. Currently there are no vaccines licensed against S. Paratyphi A. The Finlay Institute of Vaccines is working on obtaining a vaccine candidate based on outer membrane vesicles (VME) against S. Paratyphi A, so it became necessary to develop a technique for the evaluation of its immunogenicity. The objective of this work was the standardization of an ELISA for the quantification of IgG antibodies against VME of S. Paratyphi A. The best conditions of this assay were determined in terms of optimum concentration of coating and working dilution of the conjugate. In addition, the interval and linearity of the curve, the intra- and inter-assay precision, the specificity and the limit of detection were defined. The calibration curve was generated with an internal standard serum and presented a good linear fit with an R² =0.98. The coefficients of variation in the intra- and interassay precision tests were in the intervals established for each one (=10 percent, =20 percent respectively). The results obtained support the use of this quantitative ELISA for the evaluation of the immunogenicity of VME formulations of S. Paratyphi A in research and development phases(AU)
Subject(s)
Humans , Animals , Salmonella paratyphi A/pathogenicity , Paratyphoid Fever/epidemiology , Salmonella paratyphi A , Enzyme-Linked Immunosorbent AssayABSTRACT
Purpose: The present study was undertaken to analyse the trend in prevalence of culture-positive typhoid fever during the last decade and to determine antimicrobial susceptibility profile of Salmonella Typhi and Salmonella Paratyphi A isolated from patients of enteric fever presenting to our hospital. Methods: All the culture-positive enteric fever cases during 2005–2016 presenting to our Hospital were included in the study. Antimicrobial susceptibility was done against chloramphenicol, amoxicillin, co-trimoxazole, ciprofloxacin, ofloxacin, levofloxacin, pefloxacin, ceftriaxone and azithromycin as per corresponding CLSI guidelines for each year. We also analysed the proportion of culture positivity during 1993–2016 in light of the antibiotic consumption data from published literature. Results: A total of 1066 strains-S. Typhi (772) and S. Paratyphi A (294) were isolated from the blood cultures during the study. A maximum number of cases were found in July–September. Antimicrobial susceptibility for chloramphenicol, amoxicillin and co-trimoxazole was found to be 87.9%, 75.5%, 87.3% for S. Typhi and 94.2%, 90.1% and 94.2% for S. Paratyphi A, respectively. Ciprofloxacin, ofloxacin and levofloxacin susceptibility were 71.3%, 70.8% and 70.9% for S. Typhi and 58.1%, 57.4% and 57.1% for S. Paratyphi A, respectively. Azithromycin susceptibility was 98.9% in S. Typhi. Although susceptibility to ceftriaxone and cefixime was 100% in our isolates, there is a continuous increase in ceftriaxone minimum inhibitory concentration (MIC)50and MIC90values over the time. The proportion of blood culture-positive cases during 1993–2016 ranged from a minimum of 0.0006 in 2014 to a maximum of 0.0087 in 1999. Conclusion: We found that the most common etiological agent of enteric fever is S. Typhi causing the majority of cases from July to October in our region. MIC to ceftriaxone in typhoidal salmonellae is creeping towards resistance and more data are needed to understand the azithromycin susceptibility.
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Objective To understand the genomic epidemiology of Salmonella paratyphi A strains circulating in Hangzhou area in recent years. Methods Next generation sequencing(NGS) technology was used to obtain genomes of 60 Salmonella paratyphi A strains isolated in Hangzhou area from 2002 to 2013. Genomes of 391 Salmonella paratyphi A strains were downloaded from the Sequence Read Archive (SRA) and Assembly database. After removing recombinations, the phylogenetic tree of all 451 genomes based on single nucleotide polymorphisms(SNP) was constructed using ATCC9150 as the reference. SRST2 and mul-tilocus sequence typing (MLST) were used to analyze sequence types(ST). The Salmonella In Silico Typ-ing Resource (SISTR) was used for core genome multilocus sequence typing (cgMLST). Resistant genes were screened out with SRST2 and BLASTN. Seven kinds of antibiotics were selected to conduct drug sus-ceptibility test in the 60 strains isolated in Hangzhou. Results A total of 19 258 SNP loci were found in 451 genomes. The average distance in the phylogenetic tree between strains was 0.007 0 and the distance less than 0.05 accounted for 96.73%, indicating a little difference in the 451 Salmonella paratyphi A ge-nomes. Fifty-eight Hangzhou strains of ST85 were highly similar in genomic sequences,which suggested that the clonal spread of ST85 strains caused the epidemic of paratyphoid A in Hangzhou area during 2002-2013. Salmonella paratyphi A strains isolated in Hangzhou were distantly related to five domestic strains (average distance:0.057),but close to 15 Yunnan strains(average distance:0.003 2) and closest to strains isola-ted in Cambodia (average distance: 0.001 8), suggesting the possibility of transnational spread of ST85 strains. Among two Hangzhou strains of ST129,HZ333 was closely related to two strains isolated in Jiangsu Province(average distance:0.009 7),suggesting the possibility of domestic transmission of ST129 strains. Except for two untyped strains,the other 58 strains were divided into nine cgMLST types. Except for 57 un-typed strains,the other 334 strains obtained from public databases were classified into 165 cgMLST types. Fifty-six out of the 60 Hangzhou strains carried the resistant gene aac6-Iy and the other four carried aac6-Iaa gene. Thirteen out of the 391 strains obtained from public databases didn′t carry resistant genes, while the other 378 strains carried the resistant gene aac6-Iy. Among the 60 Hangzhou strains,56 were sensitive to all of the seven kinds of antibiotics;three were resistant to cotrimoxazole and one was resistant to ampicillin and tetracycline. Conclusion The epidemic of paratyphoid A fever in Hangzhou from 2002 to 2013 was mainly caused by the clonal spread of ST85 strains that had the possibility of transnational spread. Some Hangzhou strains of ST129 had the possibility of domestic transmission. SNPs analysis had an advantage over pulse field gel electrophoresis(PFGE) technology in resolution and could be used to accurately trace the cause of paratyphoid A fever. Resistant gene aac6-Iy was generally carried. NGS technology had a promising prospect in the control and prevention of infectious diseases.
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Objective@#To investigate the antimicrobial resistance and pulsed field gel electrophoresis (PFGE) patterns of S.paratyphi A strains in Zhengzhou city isolated from sentinel hospitals in 2013-2015.@*Methods@#According to Salmonella molecular typing and K-B drug susceptibility testing method published by international PulseNet bacterial infectious disease monitoring network and USA Clinical and Laboratory Standards Institute (CLSI2015), we analyzed drug sensitivity and PFGE molecular characteristics of 67 S.paratyphi A strains(11 strains in 2013, 7 strains in 2014, 49 strains in 2015) isolated from blood and stool samples in two sentinel hospitals of fever with rash syndrome surveillance system established in Zhengzhou city in 2013-2015.@*Results@#The results showed 67 strains of S.paratyphi A had different levels of resistance to 13 kinds of antibiotics, 65 strains were multi-drug resistant strains (97.0%), 5 isolates were resistant to 2-3 kinds of antibiotics (7.5%), 41 isolates were resistant to 5-8 kinds of antibiotics (61.2%),11 isolates were resistant to 9-10 kinds of antibiotics(16.4%),8 isolates were resistant to 11-12 kinds of antibiotics(11.9%). 67 strains of S.paratyphi A were divided into 10 molecular patterns(PTYA1-PTYA10) by digestion with XbaⅠ restriction endonuclease and pulsed field gel electrophoresis, each pattern contains 1-48 strains with similarity ranged from 94.31%-100%. PTYA3 contained 48 strains, which was predominant band type; PTYA1, 9 contained 6 strains; PTYA 2, 4, 5, 6, 7, 8, 10 contained 1 strains among them.@*Conclusion@#The status of drug resistance of clinical isolates of S.paratyphi A in Zhengzhou city was rather serious, PFGE patterns showed diversity and dominant characteristics. The PFGE patterns of partial strains and its corresponding anti-drug spectrum have certain relevance and cluster relationship.
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Objective To analyze the molecular characteristics of Salmonella paratyphi A ( S. pa-ratyphi A) strains prevailing in Hangzhou area in recent years. Methods Pulse field gel electrophoresis ( PFGE) and multiple-locus variable-number tandem repeat analysis ( MLVA) were performed for molecular typing and epidemiological analysis of 72 S. paratyphi A strains isolated in Hangzhou area during 2002 to 2013. Results The 72 S. paratyphi A strains were divided into 11 PFGE ( by using restriction enzymes of Xba Ⅰ and Bln Ⅰ) and 6 MLVA types. Among the selected 34 variable number tandem repeat ( VNTR) sites, 4 sites (1188K, 2075K, 2201K and 4346K) showed high polymorphism, in which PFGE displayed a higher resolution than MLVA. Except for the 5 PFGE types of X4B5, X7B7, X8B8, X9B9 and X10B10, the other 6 PFGE types belonged to a same clone sharing a similarity of greater than 95%, and the S. Paratyphi A strains in that clone accounted for 93. 1% of the total strains isolated in Hangzhou. Conclu-sion The occurrence of paratyphoid A in Hangzhou area from 2002 to 2013 was mainly caused by S. para-typhi A strains belonging to the same clone. Combination of PFGE with MLVA was conducive to epidemiolog-ical investigation of paratyphoid A.
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Background: Enteric fever is classically caused by Salmonella enterica serotype Typhi. There has been increase in enteric fever cases from different parts of India caused by Salmonella enterica serotype Paratyphi A. Thereby a retrospective study was conducted to determine the rate of isolation and antimicrobial susceptibility pattern of Salmonella Paratyphi A in comparison to Salmonella Typhi. Methods: A retrospective analysis of laboratory records was carried out from January 2011-December 2014. Conventional blood culture method was used. Salmonella were confirmed by serotyping using group and type specific anti-sera. Antibiotic susceptibility was performed for ampicillin, cotrimoxazole, ciprofloxacin, chloramphenicol and ceftriaxone, using Kirby Bauers disk diffusion method. Results: Out of 258 Salmonella isolates, 127 (49.2 %) were Salmonella Typhi and 131 (50.8 %) were Salmonella Paratyphi A. Salmonella Paratyphi A cases increased from 23.4% in 2011-2013 to 91.3% in 2014. Salmonella Typhi were 98.4 % sensitive to ampicillin and ceftriaxone and 99.2% sensitive to chloramphenicol and cotrimoxazole. Only 29.9% were sensitive to ciprofloxacin. Similarly, Salmonella Paratyphi A isolates were 99.2 % sensitive to ampicillin and 100 % sensitive to cotrimoxazole and ceftriaxone and 96.9 % sensitive to chloramphenicol and only 14.5 % sensitive to ciprofloxacin.Conclusion: The present study confers Salmonella Paratyphi A as the rapidly emerging pathogen of enteric fever. The antibiogram of Salmonella Typhi and Salmonella Paratyphi A showed decreased susceptibility to fluoroquinolones and a notable decrease in the multi drug resistant strains of Salmonella isolates with re-emergence of susceptibility to first line antibiotics.
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Background: Typhoid fever is classically caused by Salmonella enterica serotype typhi.Recently the frequency of isolation of S. paratyphi A (SPA) has been increased in comparison to S. typhi in Indian scenario. Aim: To observe the rate of isolation and antimicrobial susceptibility pattern of SPA from suspected enteric fever cases attending tertiary care centres of Eastern Orissa. Settings and Design: Retrospective study Materials and Methods: 1488 blood samples were collected during different duration of fever and cultured in BACTEC blood culture system and bottles showing signal for growth were subcultured and identified as Salmonella spp. by standard procedure and mini API (Biomeriux) and antimicrobial susceptibility by disc diffusion method. Statistical Analysis: Chi square test. Results: 167 Salmonella spp. were isolated including 83.8% Salmonella paratyphi A and 16.6% S. typhi. Among them 102 were males and 65 were females with mean age of 22.7 yrs. S. paratyphi A was the predominant spp. each year but during 2008 – 2011, there was a dramatic rise (significant P value‑ 0.034). Multidrug resistance was noticed in 10.2% of the isolates. 98% of S. paratyphi A were resistant to nalidixic acid and 41% to ciprofloxacin, but the MIC of ciprofloxacin was raised between 1‑2 μgm/dl showing the relation between nalidixic acid resistance and raised MIC of ciprofloxacin. Conclusion: Nalidixic acid should be tested along with ciprofloxacin disc while testing for susceptibility and MIC of ciprofloxacin is mandatory before advocating therapy to prevent treatment failure.
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Enteric fever due to Salmonella Paratyphi A (SPA) is a global problem occurring as outbreaks at times. An unusual SPA (2,12:a:‑) variety durazzo has been reported rarely. We report an outbreak of enteric fever due to this variety affecting 43 individuals. The blood samples grew unusual mucoid, lactose non‑fermenting colonies with atypical biochemical reactions in sugar fermentation and amino acid decarboxylation. Isolates had sensitivity to ceftriaxone, chloramphenical, cotrimoxazole, intermediate susceptibility to ciprofloxacin and resistance to ampicillin and nalidixic acid. Identification was confirmed as SPA (2,12:a:‑) at the National Salmonella Centre.
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Enteric fever is a public health problem with the upsurge in the occurrence of Salmonella isolates that are resistant to ciprofloxacin. In this study, a total of 284 blood culture isolates of S. Paratyphi A were investigated. Of these isolates, 281 (98.9%) were nalidixic acid resistant. A high rate (6.3%) of high-level resistance (≥4 µg/mL) was found to ciprofloxacin. The isolates with ciprofloxacin minimum inhibitory concentrations (MICs) of ≥12 µg/mL had 4 mutations, 2 mutations within the quinolone resistance-determining region of gyrA and 2 mutations also in parC. According to the Clinical Laboratory Standards Institute 2012 MIC breakpoints, 75.0% of isolates were resistant to ciprofloxacin. Finally, 3 major pulsed-field gel electrophoresis patterns were observed among the S. Paratyphi A isolates. The spread of fluoroquinolone resistant S. Paratyphi A necessitates a change toward ‘evidence-based’ treatment for enteric fever. The research provides a perspective on the increasing prevalence of antimicrobial resistant S. Paratyphi A isolates in this region of India.
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Objective To investigate the drug tolerance and PFGE patterns of Salrnonella (S.) paratyphi A strains isolated from sentinel hospitals in Dengfeng,Henan province,during 2009-2015.Methods Venous blood samples were collected from paratyphoid patients and cultured in double phase blood culture bottle.Suspicious strains were identified and used for Salomonella.O antigen and H1/2 phase flagellum-induced serum agglutination test with API20E biochemical systems and SSI Salmonella typing sera.According to Salmonella molecular typing and K-B drug susceptibility testing method published by PulseNet China bacterial infectious disease monitoring network and USA Clinical and Laboratory Standards Institute,we analyzed the drug susceptibility and PFGE molecule characteristics of S.paratyphiA strains isolated from the patients.Results A total of126 strains of S.paratyphi A were isolated from 248 blood samples,the antigen modes of them were 1,2,12:a:-.The resistance rate of 126 strains of S.paratyphi A was 83.3％ to ampicillin;29.4％ to ceftazidime,31.2％ to cefotaxime,17.5％ to cefepime;62.6％ to nalidixic acid;19.3％ to ciprofloxacin,26.4％ to norfloxacin;22.8％ to gentamicin,47.9％ to streptomycin;19.2％ to chloramphenicol,24.2％ to methicillin benzyl ammonium,58.6％ to compound sulfamethoxazole and 46.7％ to tetracycline.The 126 strains of S.paratyphi A had different levels of resistance to 8 kinds of antibiotics,109 strains were multidrug resistant (86.5％),9 strains were resistant to 2-3 kinds of antibiotics (7.1％),76 strains were resistant to 5-8 kinds of antibiotics (60.3％),17 strains were resistant to 9-10 kinds of antibiotics (13.5％),7 strains were resistant to 11-12 kinds of antibiotics (5.6％).The 126 strains of S.paratyphi A were divided into 14 molecular patterns by digestion with Xba I and pulsed field gel electrophoresis.The antibiotics resistance to third generation cephalosporin (CAZ,CTX),one generation and three generation of quinolones (NAL,CIP,NOR) and aminoglycosides antibiotics (STR) showed an upward trend.Each pattem contained 1-98 strains with similarity ranged from 64.10％ to 100.00％.PTYA 1,6,9 and 10 were the main PFGE belt types.Conclusion The drug resistance of clinical isolates ofS.paratyphi A was serious in Dengfeng,Henan province,PFGE pattems showed a diversity,but predominant patterns could also be found.The PFGE patterns of some strains had clustering and were related with their antidrug spectrums.
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Objective To investigate the drug tolerance and PFGE patterns of Salrnonella (S.) paratyphi A strains isolated from sentinel hospitals in Dengfeng,Henan province,during 2009-2015.Methods Venous blood samples were collected from paratyphoid patients and cultured in double phase blood culture bottle.Suspicious strains were identified and used for Salomonella.O antigen and H1/2 phase flagellum-induced serum agglutination test with API20E biochemical systems and SSI Salmonella typing sera.According to Salmonella molecular typing and K-B drug susceptibility testing method published by PulseNet China bacterial infectious disease monitoring network and USA Clinical and Laboratory Standards Institute,we analyzed the drug susceptibility and PFGE molecule characteristics of S.paratyphiA strains isolated from the patients.Results A total of126 strains of S.paratyphi A were isolated from 248 blood samples,the antigen modes of them were 1,2,12:a:-.The resistance rate of 126 strains of S.paratyphi A was 83.3％ to ampicillin;29.4％ to ceftazidime,31.2％ to cefotaxime,17.5％ to cefepime;62.6％ to nalidixic acid;19.3％ to ciprofloxacin,26.4％ to norfloxacin;22.8％ to gentamicin,47.9％ to streptomycin;19.2％ to chloramphenicol,24.2％ to methicillin benzyl ammonium,58.6％ to compound sulfamethoxazole and 46.7％ to tetracycline.The 126 strains of S.paratyphi A had different levels of resistance to 8 kinds of antibiotics,109 strains were multidrug resistant (86.5％),9 strains were resistant to 2-3 kinds of antibiotics (7.1％),76 strains were resistant to 5-8 kinds of antibiotics (60.3％),17 strains were resistant to 9-10 kinds of antibiotics (13.5％),7 strains were resistant to 11-12 kinds of antibiotics (5.6％).The 126 strains of S.paratyphi A were divided into 14 molecular patterns by digestion with Xba I and pulsed field gel electrophoresis.The antibiotics resistance to third generation cephalosporin (CAZ,CTX),one generation and three generation of quinolones (NAL,CIP,NOR) and aminoglycosides antibiotics (STR) showed an upward trend.Each pattem contained 1-98 strains with similarity ranged from 64.10％ to 100.00％.PTYA 1,6,9 and 10 were the main PFGE belt types.Conclusion The drug resistance of clinical isolates ofS.paratyphi A was serious in Dengfeng,Henan province,PFGE pattems showed a diversity,but predominant patterns could also be found.The PFGE patterns of some strains had clustering and were related with their antidrug spectrums.
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Objective:To determine the prevalence of Salmonella paratyphi C phage ( SPC-P1 ) in dif-ferent Salmonella serovars and to identify the integration sites in host genome. Methods: Based on the complete genome of SPC-P1 in S. paratyphi C RKS4594, 6 pairs of primers were designed and used to amplify the fragments of SPC-P1 in 11 S. typhi, 11 S. paratyphi A, 12 S. paratyphi B and 23 S. para-typhi C strains. At the same time, 100 complete genomes of Salmonella including 20 serovars available in National Center for Biotechnology Information ( NCBI) database were downloaded and aligned by Mauve 2. 3. 1 to determine the prevalence of SPC-P1 in these serovars. Primers were designed according to the integration sites of SPC-P1 in the genome of RKS4594 , and used to amplify ten strains having SPC-P1 in the genome. The PCR products were sequenced to investigate the integration sites of SPC-P1. Results:SPC-P1 was widely distributed in S. paratyphi C genome. In the study, 14 strains had all 6 fragments and 2 strains had 3-5 fragments. All the amplified fragments showed expected sizes. In contrast, in the ge-nomes of S. typhi, S. paratyphi A and S. paratyphi B, no or only 1-2 fragments could be amplified, and the sizes were smaller than expected. The results from Mauve showed that only in the genome of S. choleraesuis, which was a close relative of S. paratyphi C, there existed an almost complete genome of SPC-P1. The insertion site of SPC-P1 in all the ten S. paratyphi C strains tested was between pgtE and yfdC genes. Conclusion:SPC-P1 is a unique virulence factor of S. paratyphi C. It may play roles in the host range and pathogenicity of S. paratyphi C.
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Objective To develop an up-converting phosphor technology based lateral flow (UPT-LF) assay for rapid detection of Salmonella paratyphi A, S.paratyphi B, Escherichia coli O157 ∶H7 and Vibrio parahaemolyticus. Methods With up-converting phosphor nano-particles ( UCP-NPs ) as the bio-marker, four double-antibody-sandwich mode based UPT-LF strips for detecting the above mentioned four pathogens were prepared respectively and their sensitivi-ty, accuracy, linearity and specificity were evaluated .Furthermore, the feasibility of detecting bacteria in food samples was evaluated by different food samples artificially contaminated with less than 10 CFU target pathogens .Results The sensitivi-ty of UPT-LF assays for four pathogens was 105 ~106 CFU/ml with excellent specificity .The four strips had a good linear response with the linear fitting coefficient of determination (r) for each target pathogen ranging from 0.985 to 0.996.The positive rate of detecting pathogens from samples was acceptable .Conclusion The four developed UPT-LF strips provide a new choice for rapid , specific and sensitive and quantitative detection of S.paratyphi A , S.paratyphi B, E.coli O157∶H7 and V.parahemolyticus.
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Background- Systemic Lupus Erythematosus (SLE) is an inflammatory and multisystem autoimmune disorder. Patients of SLE are at increased risk of infections owing to underlying immunological derangements and to the use of therapeutic regimens like immunosuppressive agents. Among the bacterial infections presenting as bacteremia in these patients, non typhoidal and typhoidal salmonellosis are commonly encountered. We report a rare case of Salmonella Paratyphi B bacteremia in a patient with juvenile onset SLE on treatment with corticosteroids.
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Objective To construct a fusion gene (h1a-spaO) encoding H1a-SpaO protein of Sal-monella paratyphi A ( S.paratyphi A) and to express it in prokaryotic expression system , then to further ana-lyze the immunoprotective effects of the expressed protein rH 1a-SpaO.Methods The h1a-spaO fusion gene formed from separate h1a and spaO genes was amplified by PCR using flexible peptide sequence-containing linking primers and then sequenced after T-A cloning.A prokaryotic expression system for expressing h1a-spaO fusion gene was constructed by using the genetic engineering technique .The expressed protein rH1a-SpaO was examined by SDS-PAGE.The antigenicity and immunoreactivity of rH1a-SpaO protein were deter-mined by Western blot assay .The ability of rH1a-SpaO antiserum agglutinating S.paratyphi A strains was detected by micro-Widal′s test.The immunoprotective effects of rH 1a-SpaO against the lethal dose challenge of S.paratyphi A strains were analyzed in a mouse model and that were compared with those by using equal dose of individual recombinant protein H1a and SpaO (rH1a and rSpaO) as the immunogens, respectively. Results The h1a-spaO fusion gene was 100%identical with the individual h1a or spaO gene in nucleotide and amino acid sequences .The constructed prokaryotic expression system could express the recombinant pro-tein rH1a-SpaO with an advantage of high efficiency .rH1a-SpaO protein was able to react with rH 1a or rSpaO antiserum.Moreover, rH1a-SpaO antiserum also could efficiently recognize rH 1a and rSpaO as well as agglutinate Salmonella paratyphi A strains by binding with H-antigen.The immunoprotective rate (93.3%) in mice pre-immunized with 100 μg of rH1a-SpaO protein was significantly higher than that in those pre-immunized with equal dose of rH1a (60.0%) protein or rSpaO protein(53.3%) (P<0.05).Conclusion The recombinant fusion protein rH 1a-SpaO showed more stronger immunoprotective function than the individ-ual rH1a or rSpaO protein , which could be used as an effective antigen for the development of bi -valent para-typhoid A vaccine or typhoid/paratyphoid capsular polysaccharide-protein combined vaccine .
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Objective To determine the distribution and sequence conservation of outer membrane protein X (ompX) gene in Salmonella paratyphi A isolates as well as the immunogenicity and irnmono-protection of ompX gene products.Methods OmpX gene in Salmonella paratyphi A isolates was detected by PCR and the amplification products were sequenced after the T-A cloning process.OmpX gene product was expressed with E.coli expression system and the expressed rOmpX was extracted by Ni-NTA affinity chromatography.SDS-PAGE and Bio-Rad Gel Image Analyzer were applied to examine the expression and yield of rOmpX.Both antigenicity and immune-reactivity of rOmpX were detected by immune-diffusion test,ELISA and Western blot assay.The immuneprotective effect of rOmpX against infection of Salmonella paratyphi in mice was determined and the agglutinative titers of sera from rOmpX-immunized mice was measured by micro-Widal' s test.Results All the tested Salmonella paratyphi A isolates had ompX gene with high nucleotide or amino acid sequence identity (99.2％-100.0％ or 98.4％-100.0％).When rOmpX was induced to rabbits to produce high level antibody and combined with antiserum against whole cell of Salmonella paratyphi A,the results displayed a positive Western hybridization signal.Results from ELISA demonstrated that 95.6％ (65/68) of the serum samples from paratyphoid-A patients were positive on rOmpX antibody.Mice that were immunized with 100 μg or 200 μg rOmpX displayed an immune-protective rate of 93.3％ (14/15) or 100.0％ (15/15).Sera from those rOmpX-immunized mice provided 1 ∶ 10-1 ∶ 40 agglutination titers in both H antigens of Salmonella paratyphi A and Salmonella typhi.Conclusion The recombinant expression product of ompX gene could be used as a candidate antigen for developing genetic engineering vaccines against Salmonella paratyphi A infection.
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ObjectiveTo construct ompW- and ompW+ mutants of Salmonella paratyphi A with λRed system,and then study the function of the gene preliminarily.Methods Homologous regions were amplified from the genome Salmonella paratyphi A 50973,and then connect with kana fragment from plasmid pET22b-kan to construct a recombinant vector.The resultant fragments were amplified and transferred into 50973 with the help of λRed system after its concentration.Then the ompW- mutant was obtained PCR identification.Connect the recombinase expression plasmid pACU184 with full fragment of ompW regulatory region and coding region,then transfer the connection product into the mutant,the ompW+ mutant was obtained after double digest identification.Full cells of the wild,ompW- and ompW+ mutants were samples for SDS-PAGE and Western blot to detect the expression of protein OmpW.Biochemical identification of wild strain and mutant strains was conducted,so did the growth curves of the wild and the ompW- mutant.Choose BALB/c mice as a model to determine median lethal dose LD50 of the wild and mutant strains in order to observe the correlation between ompW gene and bacterial virulence.ResultsompW gene was knocked out in Salmonella paratyphi A 50973,also the ompW+ mutant was constructed; The wild and ompW+ mutant express the protein OmpW,while the ompW- mutant lost the protein.Each of the wild and mutant strains was Salmonella paratyphi A,and no obvious difference could be observed for their growth curves.LD50 for each strain was also similar.Conclusion The ompW gene has no correlation with the virulence in S.paratyphi A 50973,but the contribution of the mutants made an important foundation for the further study of functions of the gene ompW in Salmonella paratyphi.