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Aim: The present study was conducted to characterize VRSA isolates on the basis of pulsed field gel electrophoresis (PFGE) and the presence of spa gene, recovered from different Doon Valley Hospitals.Methodology: Six VRSA isolates were analyzed using PFGE and spa typing. spa gene coded Protein A was used as a genetic marker for the characterization of Staphylococcus aureus isolates. Dendrogram were constructed on the basis of unweighted pair group method with arithmetic means (UPGMA method) for clusters analyses.Results: Dendrogram finally showed two major banding patterns at about 85% similarity designated as PFGE type A and PFGE type B exhibiting differences of 4-6 bands. The length of spa gene varied from 1200 to 1500 bp, showing variation in length. The most prevalent length was 1200bp.
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Objective Acinetobacter baumannii (A. baumannii) is a commonly infective bacterium in the hospital. This study aims to analyze its molecular epidemiological characteristics, detect the carrying rate of efflux pump and regulatory protein genes, and investigate the effects of tigecycline on the efflux pump and expression of regulatory protein genes. Methods A total of 183 A. baumannii strains were collected from inpatients of the affiliated hospital of Jiangsu University from May 2017 to March 2019. They were divided into an antimicrobial-resistant group (one or more antimicrobial-resistant strains, 139 strains) and a sensitive group (the drugs in the drug sensitivity test were all non-resistant strains, 44 strains). Repeated sequence PCR was used for homology analysis of the strains, and pulse-field gel electrophoresis (PFGE) was used as the gold standard for homology analysis to verify and compare some strains. PCR was used to detect the occurrence of drug resistance-related genes. Based on homology analysis, efflux pump carrying rate detection and antibiotics sensitivity test results, 6 clinical strains carrying all efflux pump genes but different resistance phenotypes were selected as experimental strains, including sensitive strains (SAB), the multidrug resistance strain (MDRAB) and the extensively drug-resistant strain (XDRAB). All strains were induced in vitro with the minimum inhibitory concentration (MIC) of tigecycline. The induced strains were categorized as induction group, and the same strains cultured in LB agar without tigecycline was used as a control group. MIC was used to analyze the tigecycline susceptibility, and RT-qPCR was used to detect the gene expression of efflux pumps, such as TetB, AbaQ and regulatory proteins (AdeS and BaeS), in drug-resistant strains. Results Homology analysis showed that there were 45 clonal groups in the detected clinical isolates, with no obvious outbreak of epidemic clonal groups. Efflux pumps and regulatory proteins were widely distributed in the clinical isolates, and the expression of AdeB, TetB, AbeS, AdeS in MDRAB and XDRAB is significantly higher than that insensitive group SAB. Continuous in vitro induction with tigecycline could increase the antimicrobial resistance of some clinical strains and even significantly increase the expression levels of efflux pumps and regulatory proteins. Conclusion A. baumannii is widely distributed in the clinic, and efflux pumps and regulatory proteins might play an important role in drug resistance process. The unreasonable use of tigecycline could enhance the tolerance of A. baumannii by up-regulating the expression of some bacterial efflux pumps.
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Objective To study the serotype distribution characteristics and genotypes of Salmo-nella strains isolated in Yunnan Province from 2015 to 2017. Methods Automatic microbiological identifi-cation system and mass spectrometer were used to identify Salmonella strains. Their serotypes were detected using the White-Kauffmann-Le Minor (WKL) scheme based on serological detection. Genotyping was car-ried out by referring to the molecular typing method of Salmonella serotype pulse field gel electrophoresis (PFGE) in PulsenetChina. Cluster analysis was performed with Bionumerics (7.6). Results A total of 408 strains of Salmonella were detected in food and patients in Yunnan Province form 2015 to 2017,belong-ing to 70 serotypes. Thirty-four Salmonella derby strains were detected in food,accounting for 19.10% of all Salmonella strains detected in food. Among the Salmonella strains detected in patients,71 were Salmonella enteritis and 67 were Salmonella typhimurium,accounting for 30.34% and 27.63%, respectively. Results of PFGE revealed that Salmonella derby and Salmonella typhimurium were polymorphic,and Salmonella en-teritis had obvious advantages PFGE band patterns. No obvious time or geographical aggregation was found in the PFGE bands of the three Salmonella species. Conclusion Seventy Salmonella serotypes had been iden-tified in Yunnan Province by 2017. Salmonella derby was the predominant serotype detected in food, while Salmonella enteritidis and Salmonella typhimurium were the predominant serotypes in patients. These three Salmonella species caused sporadic infections in Yunnan Province.
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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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Objective To test the pathogeny of Vibrio cholerae strains of the epidemic in Jinzhou of Hubei Province.Methods Traditional methods of biochemistry,immobihzation test,string test and typing of blood serum were used to test the 6 strains isolated.The virulence gene which was cholera enterotoxin (ct) was detected by PCR.The whole genome DNA finger print of confirmed strains was analyzed by pulsed field gel electrophoresis (PFGE) after digestion respectively with two enzymes Not Ⅰ and Sfi Ⅰ.The DNA fingerprints were analyzed for clusters.Results The 6 strains of Vibrio cholerae were all O139 by traditional laboratory tests;virulence gene was detected in all 6 strains.The banding pattern was the same in the two maps of PFGE.The results of cluster analysis showed that the similarity coefficient of the six strains was 100％.Conclusion The epidemic of Vibrio cholerae is caused by the same pathogenic bacterium which is O139.
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Objective To investigate the pathogenic characteristics of Vibrio cholera strains isola-ted from Hubei province in 2012 , and to identify the source of infection by analyzing their genetic correla-tions.Methods The biochemical identification , toxin gene detection and drug susceptibility test were car-ried out to analyze a total of 35 Vibrio cholera strains isolated from three epidemic areas .Comparison of ge-nomic DNA fingerprints and cluster analysis among isolates of Vibrio cholera was conducted by using pulsed-field gel electrophoresis ( PFGE ) .Results All of the 35 strains were Vibrio cholera O139 , of which 71.42%were toxic strains.The drug resistance rates of Vibrio cholera strains to tetracycline, cotrimoxazole and rifampincin were 57.14%, 88.57%and 80.00%, respectively.Analysis of genomic DNA fingerprints of the isolates showed highly similar with similarity values ranging from 80%-100%.Most of the strains iso-lated from the same epidemic area fell into the same one cluster with 100% homology in genome Only a strain isolated from turtle in Jingzhou area was belong to a different cluster .Conclusion The Vibrio cholera O139 strains were the dominant strains causing the outbreaks of cholera in Hubei province in 2012 .Most of them were toxigenic strains .A large majority of the strains had developed resistance to tetracycline , cotri-moxazole and rifampincin , but all strains showed high susceptibility to ceftriaxone and imipenem .Vibrio cholera strains isolated from the same epidemic area were mainly belonged to the same one cluster , sugges-ting the same source of infection .However, the strains varied among different epidemic area .Follow-up in-vestigations of three outbreaks of cholera in this study were all associated with food infection .Therefore , more attention should be paid to food sanitation and safety measurement .Although a non-toxigenic strain iso-lated from turtle was not associated with the epidemic of cholera , surveillance for seafood and aquatic prod-ucts would still be necessary .
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<p><b>OBJECTIVE</b>To optimize the performance of Pulsed-Field Gel Electrophoresis (PFGE) for the comparison of inter-laboratory results and information exchange of Borrelia burgdorferi subtyping.</p><p><b>METHODS</b>A panel of 34 strains of B. burgdorferi were used to optimize PFGE for subtyping. In order to optimize the electrophoretic parameters (EPs), all 34 strains of B. burgdorferi were analyzed using four EPs, yielding different Simpson diversity index (D) values and the epidemiological concordance was also evaluated.</p><p><b>RESULTS</b>The EP of a switch time of 1 s to 25 s for 13 h and 1 s to 10 s for 6 h produced the highest D value and was declared to be optimal for MluI and SmaI PFGE of B. burgdorferi. MluI and SmaI were selected as the first and second restriction enzymes for PFGE subtyping of B. burgdorferi according to discrimination and consistency with epidemiological data.</p><p><b>CONCLUSION</b>PFGE can be used as a valuable test for routine genospecies identification of B. burgdorferi.</p>
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Animals , Humans , Rats , Bacterial Proteins , Metabolism , Bacterial Typing Techniques , Borrelia burgdorferi , Classification , Genetics , DNA, Bacterial , Metabolism , Deoxyribonucleases, Type II Site-Specific , Metabolism , Electrophoresis, Gel, Pulsed-Field , IxodesABSTRACT
Objective To determine the molecular characteristics of predominant Salmonella typhi and Salmonella paratyphi A strains prevalent in Hangzhou area from 2002 to 2008.Methods Pulse field gel electrophoresis (PFGE),multi-locus variable-number tandem repeat analysis (MLVA) and multi-locus sequence typing (MLST) were applied for typing as well as analysis of the molecular characteristics of 31 S.typhi isolates and 404 S.paratyphi A isolates from Hangzhou area during 2002 to 2008.Results The 404 S.paratyphi A isolates could be divided into six PFGE types (P1-P6).99.0％ of the S.paratyphi A isolates (400/404) belonged to the same one clone family (P1 and P2 types),in which P1 strains occupied 93.3％ (373/400) of the isolates.The 31 S.typhi isolates displayed a high diversity,which could be classified into 14 PFGE types,28 MLVA types with 90.3％ resolving power and 3 MLST types.The S.typhi strains prevalent in Hangzhou area were similar to those in Southeast Asia but different from those in Europe.The variable number tandem repeat (VNTR) sites with high polymorphism,TR1,TR2 and Sal02,could be used to the markers for diagnosis of S.typhi isolates in the area.The MLST types of 31 S.typhi isolates included all the three types currently found in the world but the ST2 type of S.typhi strains was predominant (23/31,74.2％).Conclusion The paratyphoid A prevalence in Hangzhou area in the recent years is caused by infection of the same clone family of S.paratyphi A whereas the S.typhi strains prevalent in the area display a high diversity.
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El objetivo de este trabajo fue confirmar por Electroforesis de Campo Electrico Pulsado (PFGE) que la Salmonella aislada del alimento implicado en el brote (queso blanco) fue la responsable del evento.La muestra de queso blanco presento elevado recuento de coliformes, E. coli y S. aureus,ademas, presencia de Salmo nella spp., lo que indico condiciones sanitarias inadecuadas y posible contaminacion de origen fecal. Para la confirmacion de las cepas sospechosas de Sal monella spp, aisladas de los pacientes y del alimento, se utilizaron tecnicas bioquimicas convencionales, la serotipificacion se realizo siguiendo el esquema de White-kauffmann-LeMinor y la sensibilidad a los antimicrobianos (Ampicilina, Trimetroprim-Sulfametoxazole, Ciprofloxacina, Amoxicilina, Ac.Clavulanico, Cloranfenicol, Ceftriaxone y Tetraciclina) por la tecnica kirby-Bauer. Las cepas bacterianas de Salmonella spp aisladas fueron identificadas como Salmonella Javiana [1,9,12:l, z28:1,5] y resultaron sensibles a todos los antibioticos probados.La Tipificacion Molecular de las cepas, se realizo por PFGE, se gun protocolo estandarizado de PulseNet para Salmonella y el analisis de los patrones se estudio utilizando el programa BioNumerics, version 4.0, lo cual mostro que los aisla dos de Salmonella Javiana procedentes tanto de pacientes como del alimento tenian identico patron de restriccion, en tamano y numero de fragmentos. La ocurrencia de un patron unico de PFGE (Coeficiente de similitud 100%) entre los aislados permitio demostrar que el queso contaminado con Salmonella Javiana fue el responsable del brote familiar.
The aim of this work was confirmation through Pulse Field Gel Electrophoresis (PFGE) that Salmonella isolated from the food implicated in the outbreak (white cheese) was responsible for the event. The white cheese sample showed a high count of Coli orms,f E. coli and S. aureus. Moreover, Salmonella was pre ent,s which indicated inadequate sanitary conditions and possible fecal contamination. The suspected Salmonella strains isolated from patients and the food sample, were confirmed using conventional biochemical techniques, serotyping according to the White-kauffmann-Le Minor scheme and antibiotics sensibility (Am picillin, Trimetroprim-Sulfametoxazole, Ciprofloxacin, Amoxicillin, Clavulanic Acid, Cloranfenicol, Ceftriaxone and tetraciclin) following kirby-Bauers technique. The Salmonella strains were identified as Salmonella Javiana [1,9,12:I, z28:1,5]and were sensitive to all the antibiotics tested. The molecular typing of the strains was performed using PFGE, according to the PulseNet standardized protocol for Salmonella. The pattern analysis was studied using Bionunella merics program, version 4.0, which showed that the Sal monella Javiana isolates from patients as the food sample had an identical restriction pattern in size and fragments number. The incidence of a unique pattern of PFGE (similarity coefficient 100%) between isolates demonstrated that the cheese contaminated with Salmonella Javiana was responsible for the family outbreak.
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Humans , Male , Female , Salmonella/classification , Food Contamination/prevention & control , Epidemiology , Electrophoresis/instrumentation , Salmonella/pathogenicity , Public Health , Molecular Typing/methodsABSTRACT
Las infecciones ocasionadas por Streptococcus pneumoniae constituyen un problema de salud pública. En nuestro país existe escasa información sobre aislados de procesos bacteriémicos en población adulta. Se estudió la susceptibilidad, serotipos y relación clonal de 56 aislados de S. pneumoniae desde hemocultivos, entre enero 2005 y agosto 2006, de pacientes adultos de la intercomuna Concepción-Talcahuano, Región del Bío-Bío, Chile. Se encontró resistencia a tetraciclina (21,4 por ciento), cotrimoxazol (18 por ciento), eritromicina (18 por ciento), cloranfenicol (7 por ciento) y a penicilina en un solo aislado procedente de un foco meníngeo (2 por ciento). La totalidad mostró susceptibilidad a cefotaxima, levofloxacina, moxifloxacina y vancomicina. Se demostró una amplia variedad de serotipos capsulares, con predominio de los serotipos 1, 5, 23F, 7F y 3. El análisis de macrorestricción y electroforesis en campo pulsado reveló 31 patrones electroforéticos con 12 grupos clona-les, descartando un clon predominante. De acuerdo a los resultados, al menos 80 por ciento de los serotipos de aislados de S. pneumoniae de procesos bacteriémicos están incluidos en la vacuna comercial disponible.
Streptococcus pneumoniae infections constitute a public health problem. In our country there is scarce information regarding isolates from bacteraemic episodes in adult population. The antibiotic susceptibility, sero-types and clonal relationship of 56 isolates of S. pneumoniae from adult patients with bacteraemic infections in Concepcion-Talcahuano, Bio-Bio Region, Chile, were studied. Resistance to tetracycline (21.4 percent), trimethoprim/ sulfamethoxazole (18 percent), erythromycin (18 percent), chloramphenicol (7 percent) and 1 penicillin resistant isolate from a meningeal focus (2 percent) was found. Also, all the isolates were susceptible to cefotaxime, levofloxacin, moxifloxacin and vancomycin. A wide variety of capsular serotypes was demonstrated, with predominance of serotypes 1, 5, 23F, 7F and 3. The macrorestriction analysis by pulse field electrophoresis revealed 31 electrophoretic patterns and 12 clonal groups, discarding a predominant clone. According to the results, at least, 80 percent of the S. pneumoniae serotypes isolated from bacteraemic adult patients are included in the available commercial vaccine.
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Adolescent , Adult , Female , Humans , Male , Middle Aged , Young Adult , Anti-Bacterial Agents/therapeutic use , Drug Resistance, Multiple, Bacterial/drug effects , Pneumococcal Infections/drug therapy , Streptococcus pneumoniae/drug effects , Chile/epidemiology , Chloramphenicol/therapeutic use , Electrophoresis, Gel, Pulsed-Field , Erythromycin/therapeutic use , Microbial Sensitivity Tests , Pneumococcal Infections/epidemiology , Serotyping , Streptococcus pneumoniae/classification , Tetracycline/therapeutic use , Trimethoprim, Sulfamethoxazole Drug Combination/therapeutic use , Young AdultABSTRACT
Objective To type Klebsiella pneumonia through methods including pulse-field gel electrophoresis (PFGE) in combination with multilocus sequence typing. Methods Four selected different Eps, referring to the Standard Operating Procedure of PulseNet China, were used. The single colony of Klebsiella pneumonia was quantified after enriched culture. Embedding organisms in agarose and genome DNA were lysed with Proteinase K and then digested by restriction endonuclease Xba Ⅰ , to produce agarose gel. Fingerprint was obtained through PFGE and bands were marked with their molecular weights and then analyzed by BioNumerics software. Using MLST to analyze the strains that were highly similar, by PFGE typing Results By comparing the four results from each Eps, fk3 (switch time from 6s to 36s,total run time is 18.5 hours) seemed to be better than the others.59 strains of Klebsiella pneumonia were divided into 47 PFGE types and 19 PFGE clusters. The highly similar strains could be typed into ST-340、ST-342、ST-343、ST-344、ST-345 by MLST. Among them, ST-342、 ST-343、 ST-344、 ST-345 types were all new MLST types that were reported in China.Conclusion Highly similar Klebsiella pneumonias typed by PFGE could also be typed by MLST.
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Objective To investigate the prevalence of 16S rRNA methylases gene in imipenem-resistant Acinetobacter baumannii isolates from China. Methods A total of 342 imipenem-resistant A.baumannii isolates were collected between December 2004 and December 2005, from 25 hospitals of China. Agar dilution was used to determinate the minimal inhibitory concentration (MIC) of these isolates. The homology of these isolates was analyzed by pulse-field gel electrophoresis (PFGE). Several 16S rRNA methylase genes and carbapenemase genes were detected by PCR-based assays and PCR products were sequenced. Results The rates of resistance to ampicillin-sulbactam, cefoperazone-sulbactam, tobramycin, and minocycline were 68.0%, 54.2%, 87.4%, and 75.9%, respectively. The rate of resistance to polymyxin E was 10.8%, the lowest among the tested agents. The rates of resistance to all other tested antimicrobial agents were more than 90%. The A.baumannii isolates belonged to 29 distinct clones. Among them, 6 clones were dominant, consisting of 303 isolates in total. All isolates contained the blaOXA-51-1ike gene (blaOXA-66) and 322 isolates contained the blaOXA-23-1ike gene. PCR with the ISAbal-OXA-23-like primers generated a PCR product in 314 isolates, and PCR with the ISAbal-OXA-51-1ike primers generated a PCR product in 13 strains. 221 armA-positive isolates were identified. Conclusion Most of the imipenem-resistant A.baumannii contained blaOXA-23, with ISAbal upstream of the gene. 16S rRNA methylase gene armA was widely distributed in these isolates. The results suggested that the spread of clones played an important role in the outbreak of imipenem-resistant A. baumannii in China.
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Objective To research the distribution and molecular epidemiology of insertion sequence IS1301 in Neisseria (N.) meningitidis strains in China, so as to provide scientific and available evidence for a new method of genotyping in N.meningitidis strains with IS1301. Methods Examined the IS1301 by PCR in 219 N.meningitidis strains from 16 provinces and 3 cities during 2007 and 2008 in China, productions of amplification were sent for sequencing. The positive N.meningitidis strains were analyzed by pulse field gel electrophoresis (PFGE) and nucleic acid blotting hybridization(Southern blot) by electrophoresis. Results The positive rates with IS1301 were 15.53%, 11.11%, 20.75%, 6.17% and 28.57% for four serotypes (A, B, C, N) respectively. The sequence comparability between the amplification productions and No.Z49092.1 N.meningitidis which registered in GenBank was 94%-100%. There were two types of clusters devided by cladogram analysis. There appeared large IS1301 sequence difference between the serotype C and others. The number of IS1301 replica ranged from 6-17 per strain at least. The number of IS1301 replica changed in the same type of PFGE N.meningitidis respectively. Conclusion Typing by IS1301 combined with PFGE could comprehend the homology and genetic polymorphism of N.meningitidis epidemic strains at the molecular level.
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An electrophoretic karyotype of Acanthamoeba polyphaga has been preliminarily analysed by means of pulse field gel electrophoresis (PFGE).Ten chromosomal DNA bands are distinguishable on the gel.Using yeast (Saccharomyces cerevisiae) chromosomal DNA as size standard,we estimate the size of the chromosomes to be between about 200 kilobase pairs (kb) and 2 megabase pairs (mb).