Different Species of Salmonella spp and Shigella spp and Their Risk of Infection in N’Djamena Chad

Introduction: Salmonella and Shigella are gram-negative bacilli that are resistant to most antibiotics and play an important role in an etiology of diarrhea disease. The aim of the study was to determine the prevalence of Salmonella spp and Shigella spp isolated from stool and blood samples in the city of N’Djamena. Materials and Method: This was a prospective study conducted in the four district hospitals of N’Djamena from 14 July 2022 to 31 December 2022. A questionnaire form was drawn up to collect the information sent to the study patients. The samples were analyzed at the CHU de la Mère et de l’Enfant, Labo-Redes laboratory according to their protocols and the standard of the antibiogram committee of the French microbiology society. Results: Of the 803 biological samples analyzed, 39 were positive for Salmonella spp and Shigella spp, including 15 for Salmonella and 24 for Shigella, giving an overall prevalence rate of 4.85%. Borehole water, uncooked food and lack of access to a latrine constitute a risk of being infected by Salmonella spp and Shigella spp species. Of the 8 antibiotics tested, Salmonella spp and Shigella spp strains showed good sensitivity to nalidixic acid (100% for Salmonella and 90 for Shigella) and to ciprofloxacin (90.9% for Salmonella and 75% for Shigella). Resistance to ampicillin was found in 81.81% of Salmonella species and 78.57% of Shigella species, as was resistance to chloramphenicol (81.81% of Salmonella species and 67.85% of Shigella species). Similarly, cleanliness of the service and equipment is an essential factor in preventing Salmonella and Shigella infections.

分子信标PCR检测志贺菌ipaH基因

目的用分子信标探针PCR快速检测志贺菌属(Shigella)。方法根据GenBank上公布的福氏志贺菌M32063株ipaH基因序列,设计引物和分子信标探针,以4种细菌进行对照,进行特异性和灵敏度分析,建立Shigella的实时PCR技术快速检测,应用于食物中毒和食品检测。结果检测20个样本,Shigella呈阳性,其它呈阴性,时间约2h。结论Shigella分子信标探针技术具有快速、灵敏度高、特异性强等特点,可用于Shigella食物中毒快速诊断和食品微生物检测,为食源性疾病的早期、准确诊断提供新的检测手段。

DNA染料结合环介导等温扩增技术检测志贺氏菌死活细胞

建立了一种DNA染料(EMA)结合环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)的分析方法(EMA-LAMP),用于有效检测区分病原微生物志贺氏菌的死活细胞。基于志贺氏菌ipaH基因的六个区域设计特异性的引物,检测志贺氏菌的死活细胞。结果表明:浓度为40μg/mL的EMA能够有效抑制109CFU/mL的死细胞扩增,而对相同浓度的活菌扩增没有影响。分析表明,该方法可以有效区分细菌的死活细胞,克服了传统PCR无法区分死活细胞的弊端,同时EMA-LAMP检测方法耗时短,检测灵敏度高,是一种能够有效鉴别病原菌死活细胞的新方法。

菌株Shigella flexneri FB5响应氟磺胺草醚蛋白质组学分析

为明确氟磺胺草醚降解菌的蛋白质组学相关机理,针对实验室前期筛选得到的高效降解菌Shigella flexneri FB5,在明确菌株对氟磺胺草醚高效降解的基础上,研究进行了蛋白提取和双向电泳试验。结果发现氟磺胺草醚诱导下菌株差异蛋白点13个,使用质谱技术对它们进行鉴定。通过生物信息学比对得到其功能,并对目标蛋白的基因进行PCR扩增与测序。结果表明:研究得到氟磺胺草醚诱导下菌株两个相关基因,测序后发现基因F3序列与E.coli结合蛋白dps基因同源性是100%,基因F6序列与沙门氏菌肠溶亚种血清变型索菲亚菌S1635外膜蛋白基因和E.coli SYW004外膜蛋白基因A相似度是87%,同时对这两个基因所在家族功能进行分析,并且推测出基因的理化性质。

Multi-drug Resistance and Characteristic of Integrons in Shigella spp.Isolated from China

Objective To investigate the characteristic of integrons and the relationship between integrons and antimicrobial resistance in Shigella spp. Methods Ninety Shigella strains （83 S. flexneri and 7 S. sonnei） were isolated from the stools of patients in China. Susceptibility to 8 antimicrobials was tested for all isolated strains. PCR, RFLP and sequencing analysis of integrons were applied to all of them. Results High prevalence of multi‐drug resistance （95.6%） was identified. Of the isolates 79 （87.8%） carried integrase genes of class 1 integron （3.3%）, class 2 integron （10.0%） or both （74.4%）. No intI3 was detected in the tested isolates. The prevalence of intI2 was significantly higher in isolates with multi‐drug resistance to at least 3 antibiotics than that in isolates with resistance to 2 and less antibiotics （P0.05）. Gene cassettes dfrA17‐aadA5, dfrA12‐orfF‐aadA2 of class 1 integron and dfrA1‐sat1‐aadA1 of class 2 integron were identified. Conclusion The class 2 integron may play a role in the emergence of multi‐drug resistance in Shigella spp.

Immunoproteome analysis of soluble and membrane proteins of Shigella flexneri 2457T

AIM: To profile the immunogenic proteins of Shigella flexneri (S. flexneri) expressed during human infection using a proteomic approach. METHODS: Soluble and membrane protein extractions of S. flexneri 2457T were separated by two-dimensional gel electrophoresis (2-DE). Proteins were transferred to PVDF membrane and immunoblotted with sera from shigellosis patients. Reactive protein spots were matched to Coomassie stained gels run in parallel, cut out and trypsin digested. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS) was used to determine the peptide mass fingerprints, which were searched in the MASCOT database to identify the protein. RESULTS: A total of 8 immunoreactive proteins were successfully identified from the Coomassie stained gels in three repeats. Six of these proteins have not previously been reported as immunogenic in S. flexneri. These proteins could be potential candidates for vaccine or attenuation studies. CONCLUSION: Soluble and membrane proteins of S. flexneri 2457T have been screened by 2-DE and immunoblotting with sera from shigellosis patients. Eight proteins are identified as immunogenic.

Immunoproteomics of membrane proteins of Shigella flexneri 2a 2457T

AIM： To screen the immunogenic membrane proteins of Shigella Aexneri 2a 2457T. METHODS： The routine two-dimensional polyacrylamide gel electrophoresis （2-DE） and Western blotting were combined to screen immunogenic proteins of S. Aexneri 2a 2457T. Serum was gained from rabbits immunized with the same bacteria. Immunogenic spots were cut out from the polyacrylamide gel and digested by trypsin in-gel. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry （MALDI-TOF-MS） was performed to determine the molecular weight of peptides. Electrospray ionization （ESI-MS/MS） was performed to determine the sequences of the interesting peptides. RESULTS： A total of 20 spots were successfully identified from Coomassie brilliant blue stained gels representing 13 protein entries, 5 known antigens and 8 novel antigens. A hypothetical protein （YaeT） was detected, which might be a candidate target of vaccine. CONCLUSION： Membrane proteins of S. flexneri 2a 2457T were successfully observed by 2-DE. Several known and novel antigens were identified by mass spectrum.

Prevalence and trends of aminoglycoside resistance in Shigella worldwide, 1999-2010

Shigellosis causes diarrheal disease in humans in both developed and developing countries, and multi-drug resistance in Shigella is an emerging problem. Understanding changing resistance patterns is important in determining appropriate antibiotic treatments. This meta-analysis systematically evaluated aminoglycoside resistance in Shigella. A systematic review was constructed based on MEDLINE and EMBASE databases. Randomeffect models or fixed-effect models were used based on P value considering the possibility of heterogeneity between studies for meta-analysis. Data manipulation and statistical analyses were performed using software STATA 11.0. By means of meta-analysis, we found a lower resistance to three kinds of aminoglycosides in the Europe-America areas during the 12 year study period than that of the Asia-Africa areas. Kanamycin resistance was observed to be the most common drug resistance among Shigella isolates with a prevalence of 6.88% （95%CI： 6.36%-7.43%）. Comparison of data from Europe-America and Asia-Africa areas revealed that Shigella flexneri resistance was greater than the resistance calculated for Shigella sonnei. Importantly, Shigella sonnei has played a significant role in aminoglycoside-resistance in recent years. Similarly, data showed that resistance to these drugs in children was higher than the corresponding data of adults. In conclusion, aminoglycoside-resistant Shigella is not an unusual phenomenon worldwide. Distribution in Shigella resistance differs sharply based on geographic areas, periods of time and subtypes. The results from the present study highlight the need for con- tinuous surveillance of resistance and control of antibiotic usage.

Construction of a novel Shigella live-vector strain co-expressing CS3 and LTB/STm of enterotoxigenic E.coli

AIM: To construct and evaluate a polyvalent recombinant vaccine strain Shigella flexneri2a T32 against enterotoxigenic E.coli/(ETEC). METHODS: By using a host-plasmid balanced lethal system based on asd gene, a polyvalent recombinant strain was constructed to highly express CS3 and regularly express fusion enterotoxin of LIB subunit and mutant ST (LTB/STm) in a vaccine strain Shigella flexneri 2a T32 with specific deletion of asd gene. Fimbria CS3 was observed by immunofluorescence and electron microscopy assay. The security of LTB/STm was examined by ileal loop assay and suckling mouse assay. To evaluate this new candidate vaccine, it was compared with a previous vaccine strain in plasmid and protein level, growth assay and immunogenicity in Balb/c mice. RESULTS: The newly constructed vaccine expressed CS3 and grew better than the previously constructed vaccine except for the lower expression of LTB/STm. Serum IgG and mucosal IgA against CS3, LTB, ST, and host lipopolysaccharide (LPS) were produced after immunization of Balb/c mice by oral route with the new strain. The titers were not significantly different from the Balb/c mice with the previous strain. CONCLUSION: This novel candidate diarrheal vaccine can effectively induce serum and mucosal antibody responses against ETEC and Shigella.

Pathogenic effects of O-polysaccharide from Shigella flexneri strain

AIM To investigate the specific pathogenesis ofO-polysaccharide (O--PS) which is on the outermembrane of lipopolysaccharides (LPS) fromShigella fi^eri.METHODS The O--PS was isolated and purifiedfrom Shigella nexneri 5 MgoT by enzymatichydrolysis and gel chromatography. Effects ofO--PS were observed by in vitro experiment,(HeLa cell Culture ), and in vivo experiment(rabbit lieal loop assay).RESULTS ID vitro and in vivo e-cP6riments withthe purified O--PS from Shigells flexnefi revealedthat the O--PS alone was toxic to Hela cells andcaused mucosal inflammation and hemorrhagicexudation in lieal loop of rabbit.DISCUSSION O--PS might b6 one of the factorscausing diarrhea and its mechanism wasdifferent from endotoxin reaction of LPS. Themolecular mechanism of O-PS need furtherstudies.

Comparing invasive effects of five foodborne bacterial pathogens in human embryonic intestine 407 cells and human ileocecum HCT-8 cells

Objective: To refine the infectious doses of enteric bacterial pathogens in animal assays and vaccine clinical trials by studying the invasion kinetics of five bacterial pathogens with human intestinal cells.Methods: Utilizing in vitro cultured cell invasion assays with gentamicin-killing step,the invasive effects were analyzed in foodborne pathogens including Salmonella,Shigella, Yersinia, Escherichia coli(E. coli) O157 and opportunistic pathogens Citrobacter in human embryonic intestine 407 cells and ileocecum HCT-8 cells at multiplicities of infection(MOIs) of 0.04–4 000.00 E. coli HS served as a noninvasive control.Results: The study results showed that the bacterial invasive efficiency and the average number of internalized bacteria per host cell changed with different starting MOIs. Higher starting MOIs did not always produce more bacterial internalization. The bacterial invasion effects varied with different bacterial strains and host cell lines. E. coli O157:H7 did invade human ileocecum HCT-8 cells.Conclusions: This study shows that these bacteria possess different invasive patterns at various starting MOIs and also in different cell lines. The results could help to figure out the appropriate infectious doses of the bacteria in animal assays and in vaccine clinical trials. The bacterial invasion kinetics is also valuable in evaluating the safety and efficacy of live attenuated bacterial vaccines.

Antimicrobial resistance patterns and prevalence of integrons in Shigella species isolated from children with diarrhea in southwest Iran

Objective:To investigate the antimicrobial resistance patterns and prevalence of integrons in Shigella species isolated from children with diarrhea in southwest Iran.Methods:In this study,1530 stool samples were collected from children under 15 years with diarrhea referred to teaching hospitals in Ahvaz and Abadan,southwest Iran.Shigella spp.were identified by standard biochemical tests and PCR.The antibiotic resistance pattern of all Shigella isolates was determined by the disk diffusion method and minimum inhibitory concentration(MIC)by E-test.Results:Of 1530 stool samples,91(5.9%,91/1530)were positive for Shigella spp.the most common Shigella isolates were Shigella flexneri 47(51.6%,47/1530).Antibiotic susceptibility tests showed that the highest antibiotic resistance was related to trimethoprimsulfamethoxazole(87.9%,80/91)and ampicillin(86.8%,79/91).Multiplex PCR results revealed that 56%and 86.9%of Shigella isolates carried integron classⅠand integron classⅡgenes,respectively.None of the isolates included the integron classⅢgene.Conclusions:The high prevalence of multi-drug resistance in Shigella isolates in our area increases the concerns about dissemination of the antibiotic-resistant isolates in this bacterium.

Study on Shigella Detection by ATP Bioluminescence Magnetic Enzyme Immunoassay

[Objective] The research aimed to establish a rapid detection method for Shigella. [Method] Combining immunomagnetic separation technology with ATP bioluminescence technology, a new kind of fast and accurate ATP bioluminescence magnetic enzyme immunoassay technique for Shigella was established. [Result] Using ATP bioluminescence magnetic enzyme immunoassay technique to detect standard solution for Shigella （ATCC 25931 ）, result showed that correlation coefficient between relative light intensity detected by instrument and bacteria concentration detec- ted by culture counting method was 0.981 1. Moreover, relation curve between relative light intensity and Shigella concentration was drawn. [ Conclusion] The method had a high detection speed and accuracy, and could be used for the rapid detection of pathogen in food and environment.

In vitro activity of allicin combined with two antibiotics on intestinal Shigella

Objective: We aimed to evaluate the combined antibacterial effects of allicin in combination with levofloxacin and ceftriaxone on Shigella isolated from the intestinal tract in vitro. Materials and Methods: Using a checkerboard design, broth microdilution assay was used to test the effects of the compounds on the organism. We also determined the MIC of the two groups of antibacterial drugs against 30 strains of Shigella and calculated the fractional inhibitory concentration(FIC) index, to judge the combination effect. Result: After the combined application of allicin and ceftriaxone the MIC decreased significantly. Distribution of the FIC index was as follows: FIC ≤0.5, accounting for 10%; 0.5< FIC ≤1.0, accounting for 60%; 1 < FIC ≤2, accounting for 30%; FIC >2, percentage is zero. After combined application of allicin and levofloxacin, distribution of FIC index was as follows: FIC≤0.5, ratio is zero; 0.5< FIC ≤1, accounting for 56.7%; 1 < FIC ≤2, accounting for 43.3%; FIC >2, ratio is zero. Conclusion: After the combined use of ceftriaxone, levofloxacin, and allicin, most of the tests showed synergistic effects and additive effects on Shigella, while some of them showed no correlation and no antagonistic effect.

Emerging Trends in the Etiology and Antimicrobial Susceptibility Pattern of Enteric Pathogens in Rural Coastal India

Introduction: Gastroenteritis is recognized as a serious public health problem in India. It is a syndrome that can be caused by different bacterial, viral and parasitic pathogens. Indiscriminate use of antibacterial agents has resulted in the development of multidrug resistant organisms. A retrospective study was done to analyze the etiological spectrum of diarrhoea and to determine the antimicrobial resistance pattern of bacterial pathogens. Methodology: Fecal specimens from patients with acute or chronic diarrhoea were received prior antibiotic administration and processed for routine microscopy of parasites;culture, antimicrobial susceptibility testing and polymerase chain reaction for bacterial pathogens and latex agglutination for rotavirus. Results: Out of the 6043 stool samples collected during the period of Jan 2005-Dec 2013, 678 (11.2%) enteric pathogens were isolated. The predominant isolates were Diarrheagenic Escherichia coli (DEC) (46.3%) followed by Salmonella, Shigella, Vibrio cholerae and parasites. Certain unusual pathogens were also isolated. Among age wise distribution, Diarrheagenic Escherichia coli (DEC) mainly Enteroaggregative Escherichia coli (EAEC) was isolated from <5 years of age. The majority of other enteric pathogens were isolated from the adult population. The enteric pathogens isolated showed resistance to multiple antibiotics. The resistance pattern observed during this period showed an overall increase in resistance to the antibiotics tested. Conclusion: The present study documents the overall role of etiological agents as causes of diarrhoea and indicates that the indiscriminate use of potent antibiotics can lead to acquisition of resistance to important therapeutic agents.

Multidrug Resistant <i>Shigella</i>Associated with Class 1 Integrase and Other Virulence Genes as a Cause of Diarrhea in Pediatric Patients

Background: Shigella is one of the most serious pathogens associated with bloody diarrhea in children. The empiric antibiotic therapy of enteric illness with blood streaked stool leads to emergence of multi drug resistant (MDR) Shigella. The condition gets exacerbated by presence of integrons that facilitate the horizontal spread. Virulence genes associated with MDR Shigella modulate the patient outcome, particularly in children. Objectives: The present study was aiming at isolation of MDR Shigella from children with diarrheal sickness and characterization of those isolates as regarding presence of class 1 integrase and other virulence genes. Methods: Four hundred and ninety patients under the age of five suffering from diarrheal illness were examined for presence of Shigella in their stool specimens. MDR Shigella was determined using the antibiotic susceptibility testing by disc diffusion method;those isolates were tested for presence of class 1 integrase by PCR. Multiplex PCR assay was used to determine the presence of virulence genes, virA, ial, sen, set1A, set1B, sat, ipaBCD, ipaH and stx in the MDR Shigella isolates. Results: The isolation rate of Shigella from pediatric patients was 5.3%. Most of the isolated Shigella (57.7%) were from infants between 12 and 23 month. 73.1% of the identified Shigella were MDR. intI1 gene was present in 78.9% of MDR isolates. Muliplex PCR revealed that ipaH and ipaBCD, virA, sat, ial, set1A and set1B, sen were detected in 94.7%, 78.9%, 73.7%, 68.4%, 42.1%, 36.8% of the MDR Shigella isolates respectively. Conclusion: The MDR isolates represented a considerable percentage of Shigella detected in pediatric patients. Presence of intI1 gene in most of MDR Shigella reflects the higher possibility of resistant strains spread. Existence of a variety of virulence genes in those isolates is an important indicator of serious disease outcome.

Colloidal Gold Strip-NASBA Technique for Rapid Detection of Shigella

An isothermal amplification assay for the detection of Shigella based colloidal gold strip detection is developed. The primers designed corresponded to the ipaH gene of Shigella .The sensitivity of the colloidal gold strip-NASBA method in this study was 6.3 x 101cfu/ml. The specificity of the detection system was detected and the result showed that the NASBA method could distinguish Shigella from other germs.

Evaluation of phytochemical properties and in-vitro antibacterial activity of the aqueous extracts of leaf,seed and root of Abrus precatorius Linn. against Salmonella and Shigella

Objective:To investigate the phytochemical components of Abrus precatorius(A.precatorius) and the in-vitro susceptibility of Salmonella typhi and Shigella dysenteriae to the aqueous extracts of A.precatorius leaf,seed and root.Methods:The leaf,seed and root of A.precatorius were collected and homogenized separately after drying at 40℃ for seven days in hot-air oven.The aqueous extracts of each of the parts were prepared and subjected to phytochemical screening.Dilutions of 400,300,200,100 mg/mL,of each of the extracts were used for broth dilution in minimum inhibitory concentration(MIC) determination against clinical isolates of Salmonella typhi and Shigella dysenteriae,while 50,40,30,20,and 10 mg/mL dilutions were used for the agar diffusion test and 100 mg/mL and 10 mg/mL of gentamycin were used as controls for broth dilution in MIC determination and agar diffusion test,respectively.Results:Qualitative study reveals that tannin,saponins,alkaloids,flavonoids,terpenoids,steroids and phenols were present in all of the plant parts.The leaf has the highest quantities of tannin and phenol.The root generally showed the lowest quantity of all the compounds.The pathogens were susceptible to aqueous extracts of the leaf,stem and root of A.precatorius at 50 mg/mL.At concentrations of 40,30 and 20 mg/mL,all the aqueous extracts of A.precatorius showed variation in MIC,but produced no minimum bactericide effect upon subculture.There were variations in diameter of zone of inhibition against the organisms at lower concentrations.Conclusions:These findings suggest that A.precatorius is a valuable source of phytochemicals with promising antibacterial activity.Considering this bioactivity,A.precatorius could be probed further for toxicity,and to obtain some novel antibacterial molecules.

Anti-enteric bacterial activity and phytochemical analysis of the seed kernel extract of Mangifera indica Linnaeus against Shigella dysenteriae(Shiga,corrig.) Castellani and Chalmers

Objective:To evaluate the phytochemical and anti-bacterial efficacy of the seed kernel extract of Mangifera indica(M.indica) against the enteropathogen,Shigella dysenteriae(S.dysenteriae), isolated from the diarrhoeal stool specimens.Methods:The preliminary phytochemical screening was performed by the standard methods as described by Harborne.Cold extraction method was employed to extract the bioactive compounds from mango seed kernel.Disc diffusion method was adopted to screen antibacterial activity.Minimum inhibitory concentration(MIC) was evaluated by agar dilution method.The crude extracts were partially purified by thin layer chromatography(TLC) and the fractions were analyzed by high performance thin layer chromatography(HFTLC) to identify the bioactive compounds.Results:Phytochemical scrutiny of M.indica indicated the presence of phytochemical constituents such as alkaloids,gums, flavanoids,phenols,saponins,steroids,tannins and xanthoproteins.Antibacterial activity was observed in two crude extracts and various fractions viz.hexane,benzene,chlor of orm,methanol and water.MIC of methanol fraction was found to be(95±11.8)μg/mL.MIC of other fractions ranged from 130-380μg/mL Conclusions:The present study confirmed that each crude extracts and fractions of M.indica have significant antimicrobial activity against the isolated pathogen 5. dyserUeriae.The antibacterial activity may be due to the phytochemical constituents of the mango seed kernel.The phytochemical tannin could be the reason for its antibacterial activity.

Increased systemic RNA oxidative damage and diagnostic value of RNA oxidative metabolites during Shigella flexneri-induced intestinal infection

BACKGROUND Shigella flexneri(S.flexneri)is a major pathogen causing acute intestinal infection,but the systematic oxidative damage incurred during the course of infection has not been investigated.AIM To investigate the incurred systemic RNA oxidative damage and the diagnostic value of RNA oxidative metabolites during S.flexneri-induced intestinal infection.METHODS In this study,a Sprague-Dawley rat model of acute intestinal infection was established by oral gavage with S.flexneri strains.The changes in white blood cells(WBCs)and cytokine levels in blood and the inflammatory response in the colon were investigated.We also detected the RNA and DNA oxidation in urine and tissues.RESULTS S.flexneri infection induced an increase in WBCs,C-reactive protein,interleukin(IL)-6,IL-10,IL-1β,IL-4,IL-17a,IL-10,and tumor necrosis factorα(TNF-α)in blood.Of note,a significant increase in urinary 8-oxo-7,8-dihydroguanosine(8-oxo-Gsn),an important marker of total RNA oxidation,was detected after intestinal infection(P=0.03).The urinary 8-oxo-Gsn level returned to the baseline level after recovery from infection.In addition,the results of a correlation analysis showed that urinary 8-oxo-Gsn was positively correlated with the WBC count and the cytokines IL-6,TNF-α,IL-10,IL-1β,and IL-17α.Further detection of the oxidation in different tissues showed that S.flexneri infection induced RNA oxidative damage in the colon,ileum,liver,spleen,and brain.CONCLUSION Acute infection induced by S.flexneri causes increased RNA oxidative damage in various tissues(liver,spleen,and brain)and an increase of 8-oxo-Gsn,a urinary metabolite.Urinary 8-oxo-Gsn may be useful as a biomarker for evaluating the severity and prognosis of infection.