Evaluation of zoonotic potency of Escherichia coli O157: H7 through arbitrarily primed PCR methods

Objective: To evaluate the zoonotic potency of Escherichia coli O157:H7 through arbitrarily primed-PCR (AP-PCR) methods as one of the DNA fingerprinting methods. Methods: A total of 14 isolates consisted of 11 isolates originated from human feces with renal failure symptoms, 2 isolates originated from cattle feces, and 1 control isolate were used in this study. DNA of each isolate was extracted, and their profiles were studied by using AP-PCR method with M13 F and M13 R arbitrary primers. Results: The results founded that all of 14 isolates had similarity range from 54.6% to 88.5%. Isolates KL-106(3) and KL-55(6) originated from humans showed the degree of similarity with isolates SM-25(1) and SM-7(1) originated from cattle as high as 85% and 77%, respectively. Conclusions: The high degree of similarity between isolates originated from cattle and human indicated the high potency of zoonoses. The results also concluded AP-PCR method as a briefly fingerprinting method in order to trace the epidemiological of E. coli O157:H7.

Genotypic profiles by AP-PCR of Streptococcus mutans in caries-active and caries-free preschoolers

Streptococcus mutans, an acidogenic and aciduric microorganism that colonizes the oral cavity is recognized as the main causal agent of dental caries. Epidemiological studies have shown a strong correlation between the number of S. mutans in the oral cavity and prevalence and incidence of caries. At present, different genotypic and phenotypic methods are known to determine the profiles of settling and epidemiological distribution of S. mutans. The aim of this study was to investigate the profiles of S. mutans isolated from children with and without dental caries by using the AP-PCR (arbitrarily primed polymerase chain reaction) and api-Zym methods. In the AP-PCR method, random DNA segments of the target bacterium are amplified with single primers of arbitrary sequence. The api-Zym system (bioMerieux, Marcy-letoile, France) is a phenotypic micro-method that allows simultaneous detection of 19 enzymatic activities from bacterial inoculum. A transversal observational study was conducted, which finally included 120 3- to 5- year-old children (75 with and 45 without dental caries), who attended a preschool institution in Bogota (Colombia). S. mutans was isolated from 15 of the 45 children without dental caries (33.3%) and from 31 of the 75 children with caries (41.33%). In the 46 children, 69 S. mutans isolates were identified: 24 isolates in the 15 children without dental caries and 45 isolates in 31 children with dental caries. With api-Zym system, 36 different phenotypes were detected: 22 in the caries group and 15 in the caries-free group. The phenotype XX was present in both groups. With the AP-PCR method, 27 different fingerprinting profiles were identified: 22 for the caries group and 9 of the healthy group; the two groups of patients shared four of these genomic profiles. In conclusion, the information shows a great diversity in S. mutans genotypes and phenotypes in the population studied.

La caries dental es considerada una enfermedad infecciosa multifactorial que conlleva a la destruccion del tejido dental duro. Streptococcus mutans, un microorganismo acidogenico y acidurico que normalmente se encuentra colonizando la cavidad oral, es considerado el principal microorganismo asociado al desarrollo de esta enfermedad. Estudios epidemiologicos han mostrado una fuerte correlacion entre el numero de unidades formadoras de colonias de S. mutans en la cavidad oral y la prevalencia e incidencia de caries dental. El hecho de reconocer a S. mutans como el microorganismo cariogenico mas importante, ha conducido al diseno de medidas preventivas y de control tendientes a eliminarlo o reducir su presencia en la cavidad oral. En la actualidad se utilizan diferentes metodos fenotipicos y genotipicos para demostrar la heterogeneidad y variabilidad genetica de cepas S. mutans presentes en la cavidad oral. El objetivo de este estudio fue explorar la utilidad de la tecnica APPCR en el: 1. conocimiento del genotipo en aislamientos clinicos de S. mutans provenientes de ninos con y sin caries, y 2. en el establecimiento de diferencias en los perfiles de tipificacion en comparacion con la tecnica fenotipica Api-ZYM. En el metodo AP-PCR fragmentos del DNA de la bacteria son amplificados con primers simples que se anidan al azar. El sistema api-Zym es un micro-metodo semicuantitativo de investigacion que permite detectar rapida y simultaneamente 19 actividades enzimaticas a partir de pequenas cantidades de inoculo de la bacteria. En este estudio observacional descriptivo se incluyeron finalmente 120 ninos de 3 a 5 anos de un preescolar en Bogota (Colombia). Se encontro S. mutans en 15 de los 45 ninos sin caries dental (33.3%) y en 31 de los 75 ninos con caries (41.33%). En total se identificaron 69 aislamientos de S. mutans en los 46 ninos: 24 en los 15 ninos sin caries dental y 45 en los 31 ninos con caries dental. Con el sistema Api-Zym se determinaron 36 fenotipos: 22 en el grupo de caries y 15 en el grupo sin caries. Los dos grupos solamente presentaron en comun el fenotipo XX. Con el metodo AP-PCR se identificaron 27 perfiles, 22 en el grupo con caries y 9 en el grupo sin caries; ambos grupos de pacientes compartieron 4 perfiles genomicos. En conclusion, la informacion muestra una gran diversidad en perfiles de genotipos y fenotipos de S. mutans en la poblacion objeto de estudio, los cuales en algunos casos se complementan para establecer con claridad diferencias intra e inter-individuo.

Identificação e variabilidade genética de isolados de Colletotrichum causando antracnose em inflorescências de plantas ornamentais tropicais / Identification and genetic variability of Colletotrichum isolates causing anthracnose in inflorescence of ornamental tropical plants

A antracnose afeta a qualidade de inflorescências de plantas ornamentais tropicais, e a espécie fúngica Colletotrichum gloeosporioides tem sido relacionada a essa doença apenas por análises morfológicas. Por isso, o presente trabalho teve como objetivos identificar isolados de Colletotrichum coletados em plantas de antúrio (Anthurium andraeanum), bastão do imperador (Etlingera elatior) e helicônia (Heliconia spp.), por meio de caracteres morfológicos e reação em cadeia da polimerase (PCR), e avaliar a variabilidade genética por meio de oligonucleotídeos arbitrários (AP-PCR). Pelas características morfológicas de tamanho de conídio e de apressório, todos os isolados foram identificados como C. gloeosporioides. Um fragmento de 450pb específico para C. gloeosporioides foi amplificado em todos os isolados analisados, com exceção de C 23 e C 35. A caracterização molecular realizada com três oligonucleotídeos arbitrários ((GACAC)3, (GACA)4 e (CAG)5) possibilitou a formação de três grupos de isolados, com padrões de bandas distintos. Portanto, conclui-se que as metodologias utilizadas foram eficientes na identificação de isolados de C. gloeosporioides provenientes das espécies ornamentais avaliadas e que, nos isolados analisados, não existe relação entre a similaridade observada no padrão de bandas obtido por AP-PCR e a área de coleta ou a planta hospedeira.

Anthracnose affects inflorescences quality of ornamentals tropical plants and the fungi specie Colletotrichum gloeosporioides has been related with this disease based only on morphology. Therefore, the objectives of this research was to identify Colletotrichum isolates collected on anthurium (Anthurium andraeanum), torch ginger (Etlingera elatior) and heliconia (Heliconia spp.) plants by means of morphology and polymerase chain reaction (PCR) and also verify the genetic variability using arbitrary-primed PCR (AP-PCR). All isolates were identified as C. gloeosporioides by conidium and appressorium size. A fragment of 450bp specific for C. gloeosporioides was amplified for all isolates analyzed, except for C 23 and C 35 isolates. The molecular characterization yielded three groups of isolates with different band patterns by using (GACAC)3, (GACA)4 and (CAG)5 AP-PCR. The employed methodologies were efficient to identify the C. gloeosporioides isolates collected on ornamental plants and there isn't relation between similarity of band patterns and geographic region or plant specie on the isolates analyzed.

Biochemical and molecular characterization of Bacillus pumilus isolated from coastal environment in Cochin, India / Caracterização bioquímica e molecular de Bacillus pumilus isolado do ambiente costeiro de Cochin, Índia

Bacillus species constitute a diverse group of bacteria widely distributed in soil and the aquatic environment. In this study, Bacillus strains isolated from the coastal environment of Cochin, India were identified by detailed conventional biochemical methods, fatty acid methyl ester (FAME) analysis and partial 16S rDNA sequencing. Analysis of the data revealed that Bacillus pumilus was the most predominant species in the region under study followed by B. cereus and B. sphaericus. The B. pumilus isolates were further characterized by arbitrarily primed PCR (AP-PCR), antibiotic sensitivity profiling and PCR screening for known toxin genes associated with Bacillus spp. All B. pumilus isolates were biochemically identical, exhibited high protease and lipase activity and uniformly sensitive to antibiotics tested in this study. One strain of B. pumilus harboured cereulide synthetase gene cesB of B. cereus which was indistinguishable from rest of the isolates biochemically and by AP-PCR. This study reports, for the first time, the presence of the emetic toxin gene cesB in B. pumilus.

As espécies de Bacillus constituem um grupo diversificado de bactérias amplamente distribuídas no solo e no ambiente aquático. Neste estudo, cepas de Bacillus isoladas do ambiente costeiro de Cochin, Índia, foram identificadas através de métodos bioquímicos convencionais, análise de ésteres metílicos de ácidos graxos (FAME) e sequenciamento de 16S rDNA. A análise dos dados revelou que Bacillus pumilus foi a espécie predominante na região estudada, seguido de B. cereus e B. sphaericus. Os isolados de B. pumilus foram caracterizados através da reação em cadeia da polimerase com primers arbitrários (AP-PCR), perfil de sensibilidade a antibióticos e triagem por PCR de genes de toxinas associadas com Bacillus spp. Todos os isolados de B. pumilus foram bioquimicamente idênticos, apresentaram elevada atividade de protease e lipase e foram uniformemente sensíveis aos antibióticos estudados. Um dos isolados de B. pumilus apresentou o gene cesB de B. cereus, que não foinão distinguível dos demais isolados por testes bioquímicos nem por AP-PCR. Este é o primeiro relato da presença do gene cesB da toxina eméticaem B. pumilus.

Evaluation of genotypic diversity of Streptococcus mutans using distinct arbitrary primers

Streptococcus mutans has been considered one of the main etiological agents of dental caries and the genotypic diversity rather than its salivary counts may be considered as a virulence factor of this bacterium. For genotyping with polymerase chain reaction (PCR) with arbitrary primers, several primers have been used in order to improve complexity and specificity of amplicon patterns. Thus, the aim of this study was to evaluate the degree of agreement of genotypic identification among AP-PCR reactions performed with 5 distinct arbitrary primers of S. mutans isolated from saliva. Stimulated saliva was collected from 11 adult volunteers for isolation of S. mutans, and a total of 88 isolates were genotyped with arbitrary primers OPA 02, 03, 05, 13 and 18. Fourteen distinct genotypes were identified in the saliva samples. Most volunteers (9 out of 11) presented only one genotype. The results of the present study suggest that primers OPA 02, 03, 05 and 13 were suitable for genotypic identification of S. mutans isolates of saliva from adult volunteers.

Molecular Analysis of Clostridium difficile Isolates by Arbitrarily Primed-Polymerase Chain Reaction and Polymerase Chain Reaction-Ribotyping / 감염

BACKGROUND: Clostridium difficile is known as the major cause of nosocomially acquired diarrhea. Various phenotypic and genotypic methods have been used to subtype C. difficile strains. The purpose of the present study is to evaluate several typing methods which can be used as tools for subtyping C. difficile isolates for epidemiological studies. METHODS: In two Korean tertiary care hospitals, a total of 81 C. difficile isolates were collected from symptomatic, hospitalized patients in 1998. All isolates were examined for the release of toxin A and toxin B by PCR assay and cell culture assay. Also arbitrarily primed-PCR and PCR-ribotyping profiles were determined for the typing of C. difficile strains on a genetic level. RESULTS: The toxin B gene was detected in 65.4% (54/81) of isolates by both PCR assay and cell cultureassay. Nine types were identified with T-7 primer, and 13 types were identified with PG-05 primer in AP- PCR. Sixteen types were identified in PCR-ribotyping. When two typing methods were compared, reproducibility by PCR-ribotyping was 100%, while it was only 83% and 33% AP-PCR with primer T-7, and PG-05, respectively. The discrimination index was 0.88 for PCR-ribotyping, 0.82 for AP-PCR with primer T-7 and 0.81 with primer PG-05. CONCLUSION: These data suggest that PCR-ribotyping provides a reproducible, discriminatory, and simple alternative to conventional molecular approaches for typing strains of C. difficile.

Study of Genomic Heterogeneity of Hepatocellular Carcinomas by Using Arbitrary Primer Polymerase Chain Reaction (AP-PCR) / 한국간담췌외과학회지

BACKGROUND/AIMS: Discrimination between intrahepatic metastasis (IM) and de novo multicentric occurrence (MO) in multiple hepatocellular carcinoma (HCC) is important not only for the study of hepatocarcinogenesis but also for determination of therapeutic strategies. The purpose of this study is to evaluate the clonality of multiple or recurrent hepatocelluar carcinoma by using AP-PCR. METHODS: Paraffin-embedded blocks of 9 multiple synchronous hepatocellular carcinomas, one recurrent hepatocellular carcinoma and one combined hepatocellular carcinoma and intrahepatic cholangiocarcinoma were used. None of the tumors was larger than 3.3 cm in diameter. Microdissection was done by using sterile 27 gauge needles and microscope. Two different arbitrary primers (AR3, ZF3) were utilized in AP- PCR. The clonality of tumor was assessed by DNA band pattern (DNA fingerprint) of PCR product. RESULTS: Eight of nine multiple synchronous hepatocellular carcinomas had distinctly different DNA fingerprints. One recurrent hepatocellular carcinoma and one combined hepatocellular carcinoma and intrahepatic cholangiocarcinoma also had different DNA fingerprints. CONCLUSION: AP-PCR is a simple and very powerful method for determining the clonality of multiple hepatocellular carcinomas. The majority of multiple, small-sized hepatocellular carcinomas have different clonalities and it seems that a significant number of multiple hepatocellular carcinomas are of multicentric, de novo nature.

Molecular epidemiology profile of methicillin-resistant Staphylococcus aureus in ICU ward / 中国实用内科杂志

Objective To investigate molecular epidemiology profile of methicillin-resistant Staphylococcus aureus (MRSA) in ICU ward.Methods Twenty-five MRSA strains were typed by arbitrarily primed PCR (AP-PCR).Results Ten different AP-PCR patterns (A-G) were found among 25 MRSA strains.Most of MRSA in ICU ward were A and B pulsotype.Conclusion Hospital acquired MRSA is multi-resistant to antibiotics.A and B pulsotype MRSA outbreak occures in ICU ward.

A Surveillance on Nosocomial Acquisition of Clostridium difficile and Comparative Analysis of 3 Molecular Typing Methods including Pulsed-Field Gel Electrophoresis, Arbitrarily Primed Polymerase Chain Reaction, and Ribotyping / 감염

BACKGROUND: Clostridium difficile is a major cause of nosocomial infectious diarrhea. Nosocomial clusters of C. difficile disease have been ascribed to the transfer of the organism form patient to patient. The aim of this study was to survey the nosocomial acquisition of C. difficile infection and to evaluate the efficacy and efficiency of epidemiologic typing systems by molecular analysis of the isolates. METHODS: A surveillance study for C. difficile acquisition was performed in patients admitted to neurology ward (NW) and medical intensive care unit (MICU) in an 800-bed tertiary-care hospital from August 1998 to October 1998. Stool specimens were taken weekly for culture of C. difficile. All isolates were examined for toxin B gene by PCR assay. Three molecular typing methods, including pulsed-field gel electrophoresis (PFGE), ribotyping, and arbitrarily primed polymerase chain reaction (AP-PCR) were used to differentiate individual strains of C. difficile isolates. Their performance characteristics were compared according to the consensus guidelines by the European Society for Clinical Microbiology and Infectious Disease. RESULTS: A total of 38 C. difficile strains were isolated from 308 stool cultures. The period prevalence was 7.4/1000 patient-days and 21.2/1000 patient-days in the NW and MICU, respectively (P=0.034). The acquisition incidence of C. difficile infection was 1.85/ 1,000 patient-days and 5.33/1,000 patient-days in NW and MICU, respectively. The toxin B gene was detected in 38% (8/21) of C. difficile isolates; 62.5% from diarrheal patients and 23% from asymptomatic patients. In a comparison of the three typing systems, the typeability was 0.444 by PFGE, 0.972 by AP-PCR and 1 by ribotyping, and the discrimination index was 0.975 by PFGE, 0.810 by AP-PCR and 0.777 by ribotyping. All three typing systems were highly reproducible. AP-PCR was the least costly and most rapid method. CONCLUSION: The relatively high prevalence of C. difficile infection in the hospital might indicate a potential nosocomial spread, even though the acquisition incidence was low. AP-PCR appears to be an efficacious and efficient method for the epidemiologic study of C. difficile infection, and its suboptimal discriminative power may be enhanced by complementary PFGE.

A Surveillance on Nosocomial Acquisition of Clostridium difficile and Comparative Analysis of 3 Molecular Typing Methods including Pulsed-Field Gel Electrophoresis, Arbitrarily Primed Polymerase Chain Reaction, and Ribotyping / 감염

BACKGROUND: Clostridium difficile is a major cause of nosocomial infectious diarrhea. Nosocomial clusters of C. difficile disease have been ascribed to the transfer of the organism form patient to patient. The aim of this study was to survey the nosocomial acquisition of C. difficile infection and to evaluate the efficacy and efficiency of epidemiologic typing systems by molecular analysis of the isolates. METHODS: A surveillance study for C. difficile acquisition was performed in patients admitted to neurology ward (NW) and medical intensive care unit (MICU) in an 800-bed tertiary-care hospital from August 1998 to October 1998. Stool specimens were taken weekly for culture of C. difficile. All isolates were examined for toxin B gene by PCR assay. Three molecular typing methods, including pulsed-field gel electrophoresis (PFGE), ribotyping, and arbitrarily primed polymerase chain reaction (AP-PCR) were used to differentiate individual strains of C. difficile isolates. Their performance characteristics were compared according to the consensus guidelines by the European Society for Clinical Microbiology and Infectious Disease. RESULTS: A total of 38 C. difficile strains were isolated from 308 stool cultures. The period prevalence was 7.4/1000 patient-days and 21.2/1000 patient-days in the NW and MICU, respectively (P=0.034). The acquisition incidence of C. difficile infection was 1.85/ 1,000 patient-days and 5.33/1,000 patient-days in NW and MICU, respectively. The toxin B gene was detected in 38% (8/21) of C. difficile isolates; 62.5% from diarrheal patients and 23% from asymptomatic patients. In a comparison of the three typing systems, the typeability was 0.444 by PFGE, 0.972 by AP-PCR and 1 by ribotyping, and the discrimination index was 0.975 by PFGE, 0.810 by AP-PCR and 0.777 by ribotyping. All three typing systems were highly reproducible. AP-PCR was the least costly and most rapid method. CONCLUSION: The relatively high prevalence of C. difficile infection in the hospital might indicate a potential nosocomial spread, even though the acquisition incidence was low. AP-PCR appears to be an efficacious and efficient method for the epidemiologic study of C. difficile infection, and its suboptimal discriminative power may be enhanced by complementary PFGE.

A Surveillance Study of Cooling Tower Water of Large Buildings in Seoul City for Legionella Species with Molecular Analysis / 감염

BACKGROUND: To prevent and control legionellosis outbreaks, it is important to monitor cooling towers for Legionella and establish epidemiological markers. We determined level of contamination with Legionella of cooling tower in Seoul city, analyzed the distribution of Legionella subtypes, and evaluated molecular typing methods for discrimination power and feasibility. METHODS: Water samples from 120 cooling towers in 25 areas(Gu) of Seoul city were collected during June, 1997. Culture and duplex-PCR(polymerase chain reaction) with Southern hybridization probed with Legionella-specific genes were performed with filtered samples. Twenty-two Legionella isolates were analyzed comparatively by pulsed-field gel electrophoresis(PFGE) and arbitrarily primed(AP)-PCR using a M13 reverse primer. RESULTS: Culture and duplex-PCR with Southern hybridization were positive for Legionella in 22(18.3%) and 106(88.3%) of 120 samples, respectively, resulting in 90.8%(109/120) of contamination level. Out of 22 Legionella isolates, 17 were identified as Legionella pneumophila serogroup 1, 4 as L. pneumophila serogroup 6 and 1 as an unknown. Molecular analysis of 17 isolates of L. pneumophila serogroup 1 showed 7 subtypes by PFGE(A0 in 9 isolates; A1, 2; A2, 1; A3, 2; B, 1; C, 1; D, 1) and 5 subtypes by AP-PCR(Ia in 11 isolates; Ib, 2; Ic, 2; II, 1; III, 1). The agreement of results of both methods was 76.5%(13/17) of L. pneumophila serogroup 1 and 81.8%(18/22) of all isolates, respectively. CONCLUSION: Most of cooling towers in Seoul city were already contaminated with Legionella just before summer, requiring decontamination measures and continuous surveillance. L. pneumophila serogroup 1 was the predominant isolate with variable subtypes. The AP-PCR can be used as a rapid and reproducible screening tool in tracking legionellosis outbreak.

Differentiation of Mycobacterium tuberculosis strains by arbitrarily primed polymerase chain reaction-based DNA fingerprinting

Arbitrarily primed polymerase chain reaction (AP-PCR) method was applied to the differentiation of clinical isolates of M. tuberculosis strains. The primer which is specific to human papilloma virus (HPV) type 18 was found to be appropriate for the AP-PCR-based differentiation of M. tuberculosis isolates, since AP-PCR produced multiple polymorphic DNA bands when M. tuberculosis DNA was used as template. AP-PCR was performed using either one of the HPV type 18 primer and IS6110-specific primer (half-specific PCR, HS-PCR) or HPV type 18 primer pair only (nonspecific PCR, NS-PCR). The usefulness of these two methods in differentiating M. tuberculosis isolates, was measured by calculating dissimilarity values of 16 isolates using Cluster Analysis software. The highest dissimilarity values by HS-PCR and NS-PCR methods were 0.38 and 0.59, respectively. This suggested that NS-PCR method is better than HS-PCR method in strain differentiation. Although the dissimilarity value calculated by Cluster analysis of the standard restriction fragment length polymorphism method, in which IS6110 was used as a probe, was much more higher than the NS-PCR method, NS-PCR method using HPV 18 primers was quite useful for the differentiation of M. tuberculosis strains due to its rapidity and simplicity.

Detection, Isolation and Identification of Endophytes in Lolium perenne L / 微生物学通报

A total of 61 Strains were isolated from five perennial ryegrass(Lolium perenne L.)varieties——SR4000, CalypsoⅡ, Pinnacle, Topgun and Justus. By subculture, the stable strains were separated into four morphological groups(MGs). Based on the morphological characteristics and the results of AP-PCR, 57 strains of them were identified as Neotyphodium lolii.