Serological and Antibiotic Resistance Patterns As Well As Molecular Characterization of Vibrio parahaemolyticus Isolated from Coastal Waters in the Eastern Province of Saudi Arabia.

Vibrio parahaemolyticus belongs to the halophilic genus of Vibrionaceae family that inhabits coastal and marine environments and is a major food-borne pathogen. In the Gulf Cooperation Council (GCC) countries and Saudi Arabia in particular, there is a lack of information regarding the detection of pandemic clone or serovariants of V. parahaemolyticus pandemic clones. Here, 400 seawater samples were collected and examined for the presence of V. parahaemolyticus from 10 locations along the coast of Eastern Province in Saudi Arabia. The recovered isolates were serotyped, and studied for antimicrobial resistance, virulence genes, and markers of pandemicity using PCR and Arbitrarily primed (AP)-PCR typing patterns. All 40 isolates were tested negative for tdh, trh, and toxRS genes. Six serotypes were identified and three were clinically significant. Antibiotic susceptibility testing of isolates revealed high resistance towards penicillins, cephalosporins, and polymyxin; 60% of isolates were multi-drug resistant, whereas all isolates were susceptible to quinolones, carbapenems, sulfonamides, and tetracycline. The multiple antibiotic resistance (MAR) index among antibiotic resistance patterns of isolates revealed that 12 (30%) isolates had recorded significant MAR index higher than 0.2. AP-PCR fingerprinting could group all isolates into five distinct and identical pattern clusters with more than 85% similarity. Our findings demonstrate that pandemic serovariants of pandemic clones were not exclusively limited to strains isolated from fecal specimens of infected patients. Nine environmental strains of serotype O1:KUT, O1: K25, and O5:K17 were isolated from costal seawater, and thus the spread of these serovariants strains of pandemic clone of V. parahaemolyticus in the environment is to avoid any kind of threat to public health.

Random Amplified Polymorphic DNA (RAPD) and Derived Techniques.

Understanding biology and genetics at molecular level has become very important for dissection and manipulation of genome architecture for addressing evolutionary and taxonomic questions. Knowledge of genetic variation and genetic relationship among genotypes is an important consideration for classification, utilization of germplasm resources, and breeding. Molecular markers have contributed significantly in this respect and have been widely used in plant science in a number of ways, including genetic fingerprinting, diagnostics, identification of duplicates and selection of core collections, determination of genetic distances, genome analysis, development of molecular maps, and identification of markers associated with desirable breeding traits. The application of molecular markers largely depends on the type of markers employed, distribution of markers in the genome, type of loci they amplify, level of polymorphism, and reproducibility of products. Among many DNA markers available, random amplified polymorphic DNA (RAPD) is the simplest, is cost-effective, and can be performed in a moderate laboratory for most of its applications. In addition, RAPDs can touch much of the genome and has the advantage that no prior knowledge of the genome under research is necessary. The recent improvements in the RAPD technique like arbitrarily primed polymerase chain reaction (AP-PCR), sequence characterized amplified region (SCAR), DNA amplification fingerprinting (DAF), sequence-related amplified polymorphism (SRAP), cleaved amplified polymorphic sequences (CAPS), random amplified microsatellite polymorphism (RAMPO), and random amplified hybridization microsatellites (RAHM) can complement the shortcomings of RAPDs and have enhanced the utility of this simple technique for specific applications. Simple protocols for these techniques are presented along with the applications of RAPD in genetic diversity analysis, mapping, varietal identification, genetic fidelity testing, etc.

Epidemiological characterization, genetic alterations of Helicobacter pylori infection in chronic gastric disorder and prognostic values of heterozygosity loss in chromosome 3p.

From the literature review, there seem to be no studies conducted on infection caused by Helicobacter pylori in patients with gastric MALT lymphoma in the KSA region. The present research is an attempt to understand the prevalence of patients infected with H. pylori in the selected region and the role of allelic imbalance of chromosome 3p regions to understand the clinical manifestations and features associated with MALT lymphomagenesis. The researcher analyzed the frequency of infection in patients from the region of Saudi Arabia by examining the data collected from hospitals and biopsy tissue samples as per the recommended protocol. The endoscopic diagnosis was performed to collect biopsy samples. Histology and AP-PCR DNA fingerprinting analyses were performed from the endoscopic gastric mucosal biopsies collected from patients with associated gastric MALT lymphoma. The existence of H. pylori was examined based on the results of gastric mucosal biopsies stained with hematoxylin-eosin (H&E) and Steiner's silver stains. MALT, MALT lymphoma tissue samples and H. pylori-positive chronic gastritis were examined for LOH at chromosome 3p24 using standard procedures and techniques. The findings of the paper revealed the H. pylori was found to be positive in 17% of the cases significantly high among the age group of 31-50 years. Patients with MALT, MALT lymphoma, and H. pylori-associated gastritis presented features such as lymphocyte accumulation, vacuolation, Peyer's patch appearance, and lymphatic follicles. H. pylori were found to appear as a dense colored accumulated mass in the gastric epithelial layer. The findings from AP-PCR generated DNA fingerprints revealed intense band including two prominent bands in MALT lymphoma. Among other loci, 3p24 was the only one locus that showed high percentages of LOH as reported earlier in all cancer-related cases. The findings of this research paper empower the fact that allelic imbalances play a vital role in the development of MALT lymphoma. However, future researches should be conducted to identify the chromosome regions of the AP-PCR generated DNA fingerprints of human gastric MALT lymphoma in order to confirm this proposition.

Genetic heterogeneity among Vibrio alginolyticus strains, and design of a PCR-based identification method using gyrB gene sequence.

Vibrio alginolyticus, a pathogen among humans and marine animals, is ubiquitous in marine environments. The aims of this study were to analyze the relationships between genetic diversity and origins, and to develop new primers based on the gyrB sequence to identify V. alginolyticus isolated from various sources. To determine the genetic diversity of this bacterium, an arbitrarily primed polymerase chain reaction (AP-PCR) technique was performed on 36 strains of V. alginolyticus isolated from diarrhea patients and from diseased marine animals and environments in southern Thailand. The results showed distinct DNA fingerprints of all strains, indicating that they are genetically heterogeneous. For species-specific identification of V. alginolyticus, primers targeting the gyrB gene of V. alginolyticus were developed. Thirty reference Vibrio spp., 13 non-Vibrio spp., and 160 strains of V. alginolyticus isolated from various sources in southern Thailand were used to evaluate the specificity of these primers. Our results showed that the gyrB primers could specifically identify V. alginolyticus from all sample types. In addition, the detection limit of the PCR was at least 95 pg of DNA template. Therefore, we concluded that the newly designed gyrB primers are rapid, highly sensitive, and specific to identify V. alginolyticus isolated from various sources.

Genetic variation among Clostridium perfringens isolated from food and faecal specimens in Lagos.

BACKGROUND: Clostridium perfringens is an anaerobic Gram-positive bacterium which is commonly present in the gastrointestinal tract of man and animals and causes enteritic diseases in animals and food poisoning in humans. Previous studies have looked at the epidemiological relationship between C. perfringens isolates from outbreak source. In this study, the genetic diversity of C. perfringens strains from non-outbreak food and faecal specimens was investigated for epidemiological purposes. METHODS: We analyzed thirty-eight (38) Clostridium perfringens strains isolated from food and faecal specimens in Lagos State. Bacterial identification was done using colonial morphology, Gram stain reaction, conventional biochemical tests and PCR. Genetic analysis was performed using arbitrary primed polymerase chain reaction (AP-PCR) technique with oligonucleotide primer of random sequences (OPA-3) to determine the genetic diversity of C. perfringens. The distance between the different bands produced were analyzed using numerical taxonomy and multivariate system software (NTSYS). RESULTS: Seventeen (44.7%) C. perfringens strains showed at least one polymorphic DNA patterns when genotyped. However, this method identified polymorphisms among the C. perfringens species from which four genetic groups (1, 2, 3 and 4) were established. CONCLUSIONS: Our findings suggest that there may be faecal contamination of food products and similar clones of Clostridium perfringens may be incriminated.

Age-related prevalence and characteristics of Aggregatibacter actinomycetemcomitans in periodontitis patients living in Sweden.

Background: The presence of Aggregatibacter actinomycetemcomitans in patients with periodontitis has been extensively studied for decades. Objective: To study the prevalence of A. actinomycetemcomitans in younger and older periodontitis patients and to genetically characterize isolates of this bacterium. Design: Data from microbiological analyses of 3459 subgingival plaque samples collected from 1445 patients, 337 'younger' patients (≤35 yrs) and 1108 'older' patients (>35 yrs) during 15 years (2000-2014), has been summerized. Isolates of A. actinomycetemcomitans were serotyped, leukotoxin promoter typed (JP2 and non JP2) and arbitrarily primed PCR (AP-PCR) genotyped. The origin of the JP2 genotype detected in the study population was determined. Results: The prevalence of A. actinomycetemcomitans was higher among younger than older patients and samples from the younger patients contained higher proportions of the bacterium. Serotype b was more prevalent among younger patients and the majorty of these isolates was from the same AP-PCR genotype. The JP2 genotype was detected in 1.2% of the patients, and the majority of these carriers were of non-African origin. Conslusions: For presence and charcteristics of A. actinomycetemcomitans in clinical samples the age of the carriers were a discriminating factor. Additional, apparently non-African carriers of the JP2 genotype of A. actinomycetemcomitans were identified.

Actinomyces radicidentis and Actinomyces haliotis, coccoid Actinomyces species isolated from the human oral cavity.

There are few reports on the bacterial species Actinomyces radicidentis in the literature. In this study, putative A. radicidentis isolates were collected from 16 root canal samples from 601 examined patients. The isolates were examined by biochemical tests, 16S rRNA gene sequencing, Arbitrarily-primed (AP-) PCR, antibiotic susceptibility testing, and MALDI-TOF analyses. In parallel, two A. radicidentis reference strains and two putative A. radicidentis isolates from United Kingdom were tested. Sixteen of the 18 isolates were confirmed as A. radicidentis. The remaining two isolates, both of which were isolated from root canals (one from Sweden and the other from the UK), but were identified as Actinomyces haliotis by sequencing âˆ¼ 1300 base pairs of the 16S rRNA-gene. This isolates had a divergent, but between them similar, AP-PCR pattern, and a common distribution of sequence signatures in the 16S rRNA gene, but were not identified by MALDI-TOF. A. haliotis is a close relative to A. radicidentis, hitherto only been described from a sea-snail. The identity of A. haliotis was confirmed by a phylogenetic tree based on 16S rRNA gene sequences with species specific sequences included, and by additional biochemical tests. The examined bacteria exhibited similar antibiotic susceptibility patterns when tested for 10 separate antibiotic classes with E-tests (bioMérieux). The MIC90 for ß-lactams (benzylpenicillin and cefuroxime) and vancomycin was 0.5 mg/L, for colistin and ciprofloxacin 8 mg/mL and for the other antibiotic classes ≤ 25 mg/mL The isolation of A. haliotis from infected dental root canals cast doubt on the accepted opinion that all Actinomyces infections have an endogenous source.

Antibiotic Resistance Profiles and Genotypes of Acinetobacter baumannii Isolates and In Vitro Interactions of Various Antibiotics in Combination with Tigecycline and Colistin.

OBJECTIVES: The aim of this study was to determine the antibiotic resistance profile, clonal relation and efficacy of antibiotic combinations in nosocomial multidrug resistant (MDR) Acinetobacter baumannii. MATERIALS AND METHODS: Antibiotic susceptibilities of 84 MDR A. baumannii against tigecycline (TGC), colistin (CL), amikacin (AK), ciprofloxacin (CIP), meropenem (MR), moxifloxacin (MXF), rifampicin (RF) were determined by microdilution method. Clonal relationship was investigated by genotyping using AP-PCR and antibiotyping. Interactions of antibiotic combinations were tested against clonally unrelated strains by the checkerboard (CB) method. The efficacy of the best combinations was also assesed on a selected isolate by the time-kill (TK) method. RESULTS: CIP, RF, MXF, MR, AK resistance was found as 90.47%; 47.62%; 22.62%; 58.33%; 50% respectively; however; CL and TGC were not ascertained. The isolates were distinguished as 25 different antibiotypes and 15 varied molecular patterns. The best synergistic effect was detected in combinations of CL with RF (100%) and MR (100%), in combinations of TGC with RF (53%) against clonally unrelated 15 MDR A. baumannii isolates by the CB method. While CL-RF and CL-MR showed synergy by TK method like CB, on the other hand TGC-RF indicated additive interactions by TK. CONCLUSION: In this study, both synergy tests showed that CL in combination with RF would be a good option in MDR A. baumannii.

Characterizing Diversity of Lactobacilli Associated with Severe Early Childhood Caries: A Study Protocol.

Lactobacilli have been consistently associated with dental caries for decades; however, knowledge of this group of bacteria in the etiology of the disease is limited to quantitative elucidation. Nowadays, explicit identification of oral Lactobacillus species is possible, despite their taxonomic complexity. Here we describe a combined approach involving both cultivation and genetic methods to ascertain and characterize the diversity and abundance of the Lactobacillus population in the oral cavities of children with severe early childhood caries (S-ECC). Eighty 3- to 6-year-old children (40 S-ECC and 40 caries free) who were seeking dental care at the Pediatric Dental Clinic of Bellevue Hospital in New York City were invited to participate in this study. Clinical data on socio-demographic information and oral health behavior were obtained from the primary caregiver. The data included a detailed dental examination, children's medical history, and a questionnaire survey. Combined non-stimulated saliva and supra-gingival plaque samples were collected from each child and cultivated on selective media for quantitative measures of lactobacilli levels. The procedure for Lactobacillus species screening will include the random selection of 50 colonies per plate, extraction of DNA from each colony, and genotyping by arbitrarily primed polymerase chain reaction (AP-PCR). Each unique Lactobacillus AP-PCR genotype will be selected for taxonomic assessment by 16S rRNA gene sequencing analysis. Lactobacillus species will be identified by comparing the 16S rRNA sequences with the Ribosomal Database and the Human Oral Microbiome Database. Meanwhile, the same set of clinical samples will be independently subjected to genomic DNA isolation, 16S rRNA amplification with Lactobacillus genus-specific primers, sequencing, and taxonomic identification, both at genus and species levels with a customized pipeline. The distribution and phylogenetic differences of these Lactobacillus species will be compared between children with or without S-ECC. One of the main objectives of this study is to establish a study protocol for the identification and characterization of lactobacilli in the oral cavity. Future caries risk assessments can include lactobacilli counts (quantitative) and the presence/absence of specific cariogenic genetic signatures of a Lactobacillus species (qualitative) associated with S-ECC.

Investigation of OprD Porin Protein Levels in Carbapenem-Resistant Pseudomonas aeruginosa Isolates.

BACKGROUND: The Pseudomonas aeruginosa porin OprD is a substrate-specific porin that facilitates the diffusion of basic amino acids, small peptides, and carbapenems into the cell. OprD-mediated resistance occurs as a result of decreased transcriptional expression of oprD and/or loss of function mutations that disrupt protein activity. OBJECTIVES: In this study, we examined the level of oprD expression in P. aeruginosa clinical isolates to determine the contribution of OprD porins in carbapenem resistance. MATERIALS AND METHODS: Included strains were divided into two groups, comprised of multidrug-resistant (MDR) and isolated carbapenem-resistant (ICR) strains. The transcription product level of oprD was identified using real-time polymerase chain reaction (qPCR). RESULTS: Of the 18 clinical isolates, a decrease in the oprD level was found to be significant in 13 isolates. Nine of eighteen isolates with a significant decrease were determined in the first group and comprised MDR isolates that showed a statistically significant difference compared with the ICR group (P = 0.001). In the ICR group, oprD levels were found to be significantly low in 4 isolates. Six different patterns were determined by comparing band profiles in AP-PCR. CONCLUSIONS: Although the data support the idea that the basic mechanism of imipenem resistance could be via the loss of oprD, they do not fully explain the role of oprD and indicate that other mechanisms may play an important role. Additionally, the significant decrease in the oprD levels in MDR strains suggests that oprD also plays a role in the emergence of both carbapenem and non-carbapenem resistance.

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.

Fusarium euwallaceae sp. nov.--a symbiotic fungus of Euwallacea sp., an invasive ambrosia beetle in Israel and California.

The invasive Asian ambrosia beetle Euwallacea sp. (Coleoptera, Scolytinae, Xyleborini) and a novel Fusarium sp. that it farms in its galleries as a source of nutrition causes serious damage to more than 20 species of live trees and pose a serious threat to avocado production (Persea americana) in Israel and California. Adult female beetles are equipped with mandibular mycangia in which its fungal symbiont is transported within and from the natal galleries. Damage caused to the xylem is associated with disease symptoms that include sugar or gum exudates, dieback, wilt and ultimately host tree mortality. In 2012 the beetle was recorded on more than 200 and 20 different urban landscape species in southern California and Israel respectively. Euwallacea sp. and its symbiont are closely related to the tea shot-hole borer (E. fornicatus) and its obligate symbiont, F. ambrosium occurring in Sri Lanka and India. To distinguish these beetles, hereafter the unnamed xyleborine in Israel and California will be referred to as Euwallacea sp. IS/CA. Both fusaria exhibit distinctive ecologies and produce clavate macroconidia, which we think might represent an adaption to the species-specific beetle partner. Both fusaria comprise a genealogically exclusive lineage within Clade 3 of the Fusarium solani species complex (FSSC) that can be differentiated with arbitrarily primed PCR. Currently these fusaria can be distinguished only phenotypically by the abundant production of blue to brownish macroconidia in the symbiont of Euwallacea sp. IS/CA and their rarity or absence in F. ambrosium. We speculate that obligate symbiosis of Euwallacea and Fusarium, might have driven ecological speciation in these mutualists. Thus, the purpose of this paper is to describe and illustrate the novel, economically destructive avocado pathogen as Fusarium euwallaceae sp. nov. S. Freeman et al.

Mitochondrial DNA methylation as a next-generation biomarker and diagnostic tool.

Recent expansion of our knowledge on epigenetic changes strongly suggests that not only nuclear DNA (nDNA), but also mitochondrial DNA (mtDNA) may be subjected to epigenetic modifications related to disease development, environmental exposure, drug treatment and aging. Thus, mtDNA methylation is attracting increasing attention as a potential biomarker for the detection and diagnosis of diseases and the understanding of cellular behavior in particular conditions. In this paper we review the current advances in mtDNA methylation studies with particular attention to the evidences of mtDNA methylation changes in diseases and physiological conditions so far investigated. Technological advances for the analysis of epigenetic variations are promising tools to provide insights into methylation of mtDNA with similar resolution levels as those reached for nDNA. However, many aspects related to mtDNA methylation are still unclear. More studies are needed to understand whether and how changes in mtDNA methylation patterns, global and gene specific, are associated to diseases or risk factors.

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.

Biochemical and molecular characterization of Bacillus pumilus isolated from coastal environment in Cochin, India.

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.

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.