A comparative study of coastal and clinical isolates of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium having a versatile metabolic potential and great ecological and clinical significance. The geographical distribution of P. aeruginosahas revealed the existence of an unbiased genetic arrangement in terrestrial isolates. In contrast, there are very few reports about P. aeruginosa strains from marine environments. The present work was aimed at studying the distribution of P. aeruginosa in coastal waters along the Indian Peninsula and understanding the environmental influence on genotypic, metabolic and phenotypic characteristics by comparing marine and clinical isolates. Of the 785 marine isolates obtained on selective media, only 32 (~4.1%) were identified as P. aeruginosa, based on their fatty acid methyl ester profiles. A low Euclidian distance value (< 2.5) obtained from chemotaxonomic analysis suggested that all the environmental (coastal and marine) isolates originated from a single species. While UPGMA analyses of AP-PCR and phenotypic profiles separated the environmental and clinical isolates, fatty acid biotyping showed overlapping between most clinical and environmental isolates. Our study revealed the genetic diversity among different environmental isolates of P. aeruginosa. While biogeographical separation was not evident based solely on phenotypic and metabolic typing, genomic and metatranscriptomic studies are more likely to show differences between these isolates. Thus, newer and more insightful methods are required to understand the ecological distribution of this complex group of bacteria.

AP-PCR and Antimicrobial Susceptibility Patterns of Fusobacterium nucleatum Associated with Chronic Periodontitis among Patients at Lagos University Teaching Hospital.

Aims: To isolate, identify and evaluate the genetic diversity and antimicrobial susceptibility of F. nucleatum recovered from Nigerian patients with chronic periodontitis. Study Design: Cross-sectional design. Place and Duration of Study: Department of Medical Microbiology and Parasitology, College of Medicine, University of Lagos, between January 2007 and July 2008. Methodology: We analyzed F. nucleatum species recovered from Nigerian patients with chronic periodontitis. Bacterial identification was done using colonial morphology; Grams stain reaction, conventional biochemical tests, API 20-A and Polymerase chain reaction (PCR). The minimum inhibitory concentration (MIC) of 6 antibiotics was determined by agar dilution method on Brucella blood agar while the bacterial genetic diversity was studied using the Arbitrarily Primed-PCR (AP-PCR) method with the arbitrary primer OPA-05. The interrelationship and genetic similarity matrix among the isolates was determined and by Numerical taxonomy and multivariate analysis system (NTSYS-pc) statistical package. Results: We obtained 48 isolates of F. nucleatum from 50 Nigerian patients (28 males and 22 females) with chronic periodontitis. They were susceptible to metronidazole, clindamycin, cefoxitin, tetracycline, amoxicillin and clavulanate. One was resistant to amoxicillin (MIC >32 μg/ml) and produced β-lactamase. The isolates were further placed into five groups (A, B, C, D and E) based on their AP-PCR profile. Conclusion: The AP-PCR analysis showed heterogeneity among strains. By using APPCR, we observed a single β-lactamase producing clone resistant to amoxicillin which eventually formed a distinct group showing that such genetic difference may have contributed to the formation of a separate clone.

Genotypic diversity of S. mutans in dental biofilm formed in situ under sugar stress exposure

In situ dental biofilm composition under sugar exposure is well known, but sugar effect on the genotypic diversity of S. mutans in dental biofilm has not been explored. This study evaluated S. mutans genotypic diversity in dental biofilm formed in situ under frequent exposure to sucrose and its monosaccharide constituents (glucose and fructose). Saliva of 7 volunteers was collected for isolation of S. mutans and the same volunteers wore intraoral palatal appliances, containing enamel slabs, which were submitted to the following treatments: distilled and deionized water (negative control), 10 percent glucose + 10 percent fructose (fermentable carbohydrates) solution or 20 percent sucrose (fermentable and EPS inductor) solution, 8x/day. After 3, 7 and 14 days, the biofilms were colleted and S. mutans colonies were isolated. Arbitrarily primed polymerase chain reaction (AP-PCR) of S. mutans showed that salivary genotypes were also detected in almost all biofilm samples, independently of the treatment, and seemed to reflect those genotypes present at higher proportion in biofilms. In addition to the salivary genotypes, others were found in biofilms but in lower proportions and were distinct among treatment. The data suggest that the in situ model seems to be useful to evaluate genotypic diversity of S. mutans, but, under the tested conditions, it was not possible to clearly show that specific genotypes were selected in the biofilm due to the stress induced by sucrose metabolism or simple fermentation of its monosaccharides.

A composição do biofilme dental in situ exposto a açúcares é bem conhecida, mas o efeito dos açúcares na diversidade genotípica de S. mutans no biofilme dental ainda não foi explorada. Este estudo avaliou a diversidade genotípica de S. mutans no biofilme dental formado in situ sob frequente exposição à sacarose e seus monossacarídeos constituintes (glicose e frutose). Primeiramente, saliva de voluntários foi coletada para isolamento de S. mutans e os mesmos voluntários usaram um dispositivo intraoral palatino, contendo blocos de esmalte, que foram submetidos 8x/dia aos seguintes tratamentos: água destilada e deionizada (controle negativo), solução de glicose 10 por cento + frutose 10 por cento (carboidratos fermentáveis) e solução de sacarose 20 por cento (fermentável e indutor de PEC). Após 3, 7 e 14 dias, os biofilmes foram coletados e colônias de S. mutans foram isoladas. A técnica de reação em cadeia de polimerase usando primers arbitrários (AP-PCR) demonstrou que o genótipo salivar foi detectado em quase todas as amostras de biofilme, independente do tratamento, e parece refletir aqueles genótipos presentes em maiores proporções no biofilme. Além do genótipo salivar, outros foram encontrados nos biofilmes, mas em uma menor proporção e foram distintos entre os tratamentos. Os dados sugerem que o modelo in situ é útil para a avaliação da diversidade genotípica de S. mutans. Porém, nas condições do presente estudo, não foi possível demonstrar que genótipos específicos foram detectados no biofilme devido ao estresse induzido pelo metabolismo da sacarose ou fermentação de seus monossacarídeos.

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 control study of S.mutans genotype in the oral cavity of mothers-children pairs / 实用口腔医学杂志

Objective:To study the relationship between genetic diversity within S.mutans and dental caries.Methods:Isolates of S.mutans were obtained from 10 caries active children and their mothers,and were originally isolated on mitis-salivarious-bacitracin agar.A total of 800 isolates were biochemically confirmed as S.mutans.Chromosomal DNA was extracted from those isolates with an extraction kit.By AP-PCR,the genetic diversities of S.mutans were examined respectively in 10 caries active children,10 caries-free children,and their mothers.DNA fingerprints were obtained by electrophoresis on 15 g/L agarose gel and analyzed for genotypics similarities.Results:The number of distinct genotypes of S.mutans harbored in caries active children were,on average,greater than that presented in children without caries(P

Isolation of Candidate Tumor Suppressor Gene in Renal Cell Carcinoma / 대한임상병리학회지

BACKGROUND: Although renal cell carcinoma (RCC) is the most common malignancy originated from kidney in adults, pathogenesis of RCC remains unknown. The purpose of this work is to find tumor suppressor gene in RCC. METHODS: The arbitrarily primed polymerase chain reaction (AP-PCR) has been used to detect somatic genetic alterations in RCC. DNA fingerprints generated by single arbitrary primers were compared between normal and tumor tissues of the same individuals. AP-PCR bands showing decreased intensities in tumor tissue DNA, relative to normal, have been cloned after reamplification with the same arbitrary primer. We have performed Southern blot hybridization and DNA sequencing. RESULTS: For a given primer, at least 5 differences in band patterns between normal and tumor tissues were observed and band C was deleted in tumor tissues of clear cell type RCC. We found this band was split into 3 bands. Because band C2 was consistantly deleted in tumor tissue, we decided to clone and characterize this fragment. Partial DNA sequences of this fragment showed no homology with other genes by BLAST search. Southern blot analysis showed this fragment was deleted in 2 cases of clear cell type and 1 case of mixed cell type RCC. CONCLUSIONS: These results suggest that fragment C2 might be a candidate for novel tumor suppressor gene and loss of this fragment might be necessary for malignant development to clear cell type RCC. Further characterization of this fragment is expected to give us useful informations about RCC tumorigenesis.

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.

Molecular Determination of Dermato phyte Fungi Using Arbitrarily Prime d Polymerase Chain Reaction / 中华皮肤科杂志

Objective To study the significance of AP-PCR i n identification and subtyping of der-matophytes.Methods Using a pair of random primers,OPAA11(5' -ACCCGACCTG -3' ),and OPD18(5' -GAGAGCCAAC -3' )the DNAs of 64isolates of dermatophytes(9species of 3genera),Sporothrix schenckii and Candida albicans were amplified by AP-PCR and analyse d by electrophoresis.Results Distinct DNA band patterns were observed in diffe rent dermatophyte species.Common major DNA bands were observed in Trichophyton rubrum isolated from different areas with s train difference.Conclusion Using OPAA11and OPD18as primers,AP-PCR may be applied in the identification and subtypi ng of dermatophytes.