Arbitrarily Primed Polymerase Chain Reaction Research Articles

Strain-level detection of Fusobacterium nucleatum in colorectal cancer specimens by targeting the CRISPR-Cas region.

Fusobacterium nucleatum is associated with colorectal cancer (CRC), and identical F. nucleatum strains seemed to be detected in 75% of patients who exhibited the presence of F. nucleatum in both CRC and saliva samples in our previous study; however, the validation of strain identity and the development of a method for strain-level discrimination are required for etiological studies. We confirmed that F. nucleatum isolates obtained from CRC and saliva samples derived from patients with CRC originated from their identical strains using whole-genome sequencing. We evaluated the hypervariable region of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CRISPR-Cas) system in F. nucleatum strains isolated from the CRC and saliva specimens of CRC patients to develop a method for genotyping this bacterium at the strain level. The developed method consisted of two simple PCR steps to amplify the different lengths and sequences of the CRISPR-Cas regions from F. nucleatum strains using specific but common primers. This method could successfully detect identical strains present in both cryopreserved CRC samples and saliva obtained from the same CRC patient. Dynamic monitoring of F. nucleatum strains in saliva obtained from patients with colorectal adenoma before and after oral care showed interindividual variability in F. nucleatum strains during oral care. This study provided a simple and rapid method for comprehensively identifying F. nucleatum at the strain level in clinical samples, leading to a paradigm shift in CRC research, such as the investigation of pathogenic F. nucleatum strains and monitoring of pathogenic strains in clinical trials for preventing CRC recurrence. (This study was registered in the UMIN Clinical Trials Registry under ID UMIN000016229.) IMPORTANCE Fusobacterium nucleatum is one of the predominant oral bacteria in humans. However, this bacterium is enriched in colorectal cancer (CRC) tissues and may be involved in CRC development. Our previous research suggested that F. nucleatum is present in CRC tissues originating from the oral cavity using a traditional strain-typing method [arbitrarily primed polymerase chain reaction (AP-PCR)]. First, using whole-genome sequencing, this study confirmed an exemplary similarity between the oral and tumoral strains derived from each patient with CRC. Second, we successfully developed a method to genotype this bacterium at the strain level, targeting the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated system, which is hypervariable (defined as F. nucleatum-strain genotyping PCR). This method can identify F. nucleatum strains in cryopreserved samples and is significantly superior to traditional AP-PCR, which can only be performed on isolates. The new methods have great potential for application in etiological studies of F. nucleatum in CRC.

A system for transposon mutagenesis of Bartonella bacilliformis

Bartonella bacilliformis is the etiologic agent of Carrión's disease in South America. Lack of a system for random mutagenesis has significantly hampered research on the pathogen's molecular biology. Here, we describe a transposon (Tn)-based mutagenesis strategy for B. bacilliformis using pSAM_Rl; a Tn-mariner delivery vector originally constructed for members of the Rhizobiaceae family. Following electroporation of the vector, five candidate mutant strains were selected based on aberrant colony morphologies, and four mutations confirmed and identified using arbitrarily-primed PCR coupled with Sanger sequencing. One mutant strain, 4B2, was found to have a disrupted flgI gene, encoding the P-ring component of the flagellar motor. We therefore investigated the flgI strain's motility phenotype in a novel motility medium and found that insertional mutagenesis produced a non-motile mutant. Taken as a whole, the results show that: 1) pSAM_R1 is a practical Tn delivery vector for B. bacilliformis, 2) the plasmid can be used to create random Tn mariner mutants, 3) arbitrarily-primed PCR coupled with Sanger sequencing is a rapid and simple method for identifying and locating mutations generated by this Tn, and 4) in silico-predicted mutant phenotypes can be verified in vitro following mutagenesis. This system of Tn mutagenesis and mutation identification provides a novel and straightforward approach to investigate the molecular biology of B. bacilliformis.

Prevalence, Antibiotic Resistance, Toxin-Typing and Genotyping of Clostridium perfringens in Raw Beef Meats Obtained from Qazvin City, Iran.

Background: Clostridium perfringens is one of the highest prevailing spore-forming foodborne pathogens, which is widely distributed and causes severe disease and outbreaks in humans and animals. Raw meat and poultry are the main vehicles of this pathogen. In this study, we investigated the prevalence, antibiotic resistance pattern, toxin-encoding genes and genetic diversity of C. perfringens isolates from raw whole and minced meat samples purchased from local markets in Qazvin city, Iran (the source of beef cattle production was also located in Qazvin city, Iran). Methods: We used conventional culture-based and Kirby–Bauer disk diffusion and conventional and arbitrary primer PCR methods. Results: A total of 18 C. perfringens strains were isolated from 133 raw meat samples (13.53%). Up to 44.4 and 55.5% of these isolates were detected in raw minced and whole meat samples, respectively. We found that 72.2, 66.6, 61.1, 37.8 and 33.3% of the C. perfringens isolates were resistant to ampicillin, tetracycline, amoxicillin, ciprofloxacin and chloramphenicol antibiotics, respectively. Multidrug resistance was found in 38% of the isolates. Among the four main toxin genes evaluated, the Cpa gene was detected in all isolates, and 61.1% of the isolates were mostly recognized as type A C. perfringens. High levels of genetic diversity were observed among the isolates, and they were classified into five distinct groups. Conclusions: The isolates from whole meat samples were more resistant to antibiotics. However, toxin genes were more detected in the isolates from minced meat samples. Our findings suggest that contamination of raw meat products with multidrug resistant C. perfringens could be regarded as one of the concerning pathogens in these products. Comprehensive monitoring of C. perfringens isolates is strongly recommended.

Estimating Genotypic Diversity of Streptococcus mutans Isolated From Caries-Active and Caries-Free Individuals Among Indian Population.

PurposeTo identify the genetic characterization of S mutans strains isolated from the caries-free and caries-active population using arbitrarily primed -polymerase chain reaction (AP-PCR).Materials and methodsThirty-five subjects with a decayed missing and filled teeth (DMFT) score of 0 were allocated to the caries-free group and 35 subjects with a DMFT score greater than four were allocated to the caries-active group and salivary samples were collected. The samples were processed and the colony forming units (CFU) of S mutans were determined for all the samples. About three colonies resembling S mutans from each subject were subjected to deoxyribonucleic acid (DNA) isolation, a purification process was performed, and DNA was analyzed by AP-PCR.ResultsAmong the 210 isolates from 35 caries-active and 35 caries-free subjects that were analyzed by AP-PCR, 41.9% had a single genotype, 25.8% had two genotypes, 19.4% had three genotypes, 9.7% had four genotypes and 3.2% had more than four genotypes among the caries-free group. Among the caries-active group, 54.3% had a single genotype, 25.7% had two genotypes, 14.3% had three genotypes and 5.7% had four genotypes.ConclusionThe study concluded that different genotypic pattern was present in caries-free and caries-active subjects which indicate the occurrence of genetic polymorphism among the S mutans strains. There was no particular genotype of S mutans that was uniquely present in subjects in caries-active and caries-free individuals.

Infection control measures to stop the spread of sequence type 15 OXA-23-producing Acinetobacter baumannii in a Swedish Burn Center

ObjectiveTo describe the course of the outbreak and infection control measures to stop the spread of sequence type 15 OXA-23-producing Acinetobacter baumannii in the Burn Center of Uppsala University Hospital, between November 2014 and the end of April 2015. MethodsCompliance with hand hygiene, dress code, and cleaning routines were reviewed, the ward’s environment was systematically investigated to identify potential environmental sources. Sampling routines for A. baumannii, from patients and environment, were established, and the epidemiological relationship was analysed for all carbapenem-resistant A. baumannii isolates using arbitrarily primed polymerase chain reaction (AP-PCR) and pulsed-field gel electrophoresis (PFGE). ResultsA total of 54 patients were treated at the burn intensive care unit during the studied, approximately five months period, and an OXA-23-producing A. baumannii was isolated from nine patients (9/54, 17%), whereof two died (2/9, 22.2%). All isolates shared identical PFGE-genotype patterns and belonged to sequence type 15; AP-PCR was eligible for prompt epidemiological investigations. ConclusionsHigher awareness and increased compliance with hand hygiene and dress code as well as intensified cleaning protocols of the environment and equipment were successfully established and likely to have led to stop the spread of sequence type 15 OXA-23-producing Acinetobacter baumannii.

Origin of <i>Candida albicans</i> in Human Oral Cavity

Purpose: Candida albicans is regarded as a part of normal flora in the human oral cavity. However, it remains unclear whether the genus Candida, especially C. albicans, is an oral resident microorganism and causes marital infection or not. The purpose of the present study was to elucidate the origin of oral C. albicans by investigating the colonization and infection route to oral cavities of this organism with arbitrarily primed polymerase chain reaction (AP-PCR). Methods: After C. albicans was isolated from four subjects (average age: 42.2, range: 33 - 56), the isolations of this organism from them were performed six months later again. To investigate whether C. albicans is an oral resident microorganism, the genotype homology of each C. albicans isolates that were isolated twice from the same subjects was compared. Moreover, C. albicans was isolated from five pairs of married couples (average period of cohabitation: 12.4 years, range: 5 - 31). To investigate whether C. albicans causes marital infection, the genotype homology of C. albicans isolates that were isolated from each pair of married couples was compared. Results: AP-PCR patterns of C. albicans that were isolated from each subject at o month and after 6 months showed the identical genotypes among each individual. C. albicans isolates from five pairs of married couples showed the identical genotypes between a husband and wife of each pair on AP-PCR. Conclusion: These results indicated that C. albicans was an oral resident microorganism and caused the marital infection.

Mutans Streptococci and Lactobacilli: Colonization Patterns and Genotypic Characterization of Cariogenic Bacterial Species in American Indian Children

Aim: The purpose of this study was to investigate the presence of Streptococcus mutans, Streptococcus sobrinus, and Lactobacillus species in an American Indian population displaying a high incidence of severe early childhood caries (S-ECC) and to explore the genotypic diversity and fidelity of transmission of S. sobrinus in this population.Methods: We report here on the microbial profiles of 71 children compiled from birth to 36 months of age and initial exploration of genotypic diversity in a subset of 40 mother/child dyads. Whole mouth plaque samples were collected from mother/child dyads in a Northern Plains American Indian cohort. Mutans streptococci (MS) and Lactobacillus counts were recorded and presence/absence of S. mutans, S. sobrinus, and Lactobacillus was noted for each child. S. sobrinus was isolated and genotyped via arbitrarily primed-polymerase chain reaction (AP-PCR).Results: The children in this study were divided into caries active (CA) and caries free (CF) groups. S. mutans, S. sobrinus, and Lactobacillus species were detected more frequently in CA groups. Distribution of MS and Lactobacillus counts was significantly different between the two groups, as was the distribution of three species colonization profiles. Twelve S. sobrinus genotypes were detected in the subset of mother/child dyads. Individual participants displayed a range of 0–3 distinct genotypes while mother/child pairs displayed a range of 1–5 genotypes. Eighty-three percent of children shared at least one genotype with his/her mother.Conclusion: Caries active children in this population display different microbial profiles than the caries free children in this cohort. A small number of S. sobrinus genotypes appear to be very common in this population. A high rate of vertical transmission of S. sobrinus from mother to child is seen in these mother/child dyads.

Genotypic and phenotypic characterization of Streptococcus mutans strains isolated from patients with dental caries.

Background and Objectives:The oral cavity harbors numerous Streptococcus mutans strains which display remarkable genotypic and phenotypic diversity. This study evaluated the genotypic and phenotypic diversity of 209 S. mutans strains isolated from 336 patients with dental caries and compared with the universal reference strain, UA159.Materials and Methods:Selective cultivation on mitis-salivaries-bacitracin agar and species-specific polymerase chain reaction (PCR) was carried out to isolate and identify the 209 S. mutans isolates from 336 patients with dental caries. Arbitrarily primed polymerase chain reaction (AP-PCR), PCR amplification of specific gene, acid production and biofilm formation capacity were performed to evaluate the genotypic and phenotypic variation. Student’s t-test and Chi-square test were used for analysis of variables and a probability (P) of <0.05 was considered as significant.Results:Our study revealed a high degree of genotypic and phenotypic variability among the clinical strains. We observed significant differences in colony morphology, generation time, biofilm formation, and acid production while growing in culture medium. All the clinical isolates were able to lower pH while growing in Todd-Hewitt broth. Consistent with phenotypic variations, we also observed genotypic variation by AP-PCR and gene specific PCR. AP-PCR analysis suggested that most of the patients with dental caries have distinct type of S. mutans strains. Genes related to various two component systems were highly conserved among the isolated strains, however, bacteriocin encoding genes such as nlmAB, nlmC were absent in nearly half of the clinical isolates.Conclusion:Our results support that S. mutans clinical isolates have wide genotypic diversity and show variation in growth kinetics, acid production, acid tolerance and biofilm formation capacity and indicates the presence of diverse mechanism to initiate and establish the biofilm lifestyle which leads to tooth decay.

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.

Identification of the RNase-binding site of SARS-CoV-2 RNA for anchor primer-PCR detection of viral loading in 306 COVID-19 patients

The pandemic of coronavirus disease 2019 (COVID-19) urgently calls for more sensitive molecular diagnosis to improve sensitivity of current viral nuclear acid detection. We have developed an anchor primer (AP)-based assay to improve viral RNA stability by bioinformatics identification of RNase-binding site of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and implementing AP dually targeting the N gene of SARS-CoV-2 RNA and RNase 1, 3, 6. The arbitrarily primed polymerase chain reaction (AP-PCR) improvement of viral RNA integrity was supported by (a) the AP increased resistance of the targeted gene (N gene) of SARS-CoV-2 RNA to RNase treatment; (b) the detection of SARS-CoV-2 RNA by AP-PCR with lower cycle threshold values (−2.7 cycles) compared to two commercially available assays; (c) improvement of the viral RNA stability of the ORF gene upon targeting of the N gene and RNase. Furthermore, the improved sensitivity by AP-PCR was demonstrated by detection of SARS-CoV-2 RNA in 70–80% of sputum, nasal, pharyngeal swabs and feces and 36% (4/11) of urine of the confirmed cases (n = 252), 7% convalescent cases (n = 54) and none of 300 negative cases. Lastly, AP-PCR analysis of 306 confirmed and convalescent cases revealed prolonged presence of viral loading for >20 days after the first positive diagnosis. Thus, the AP dually targeting SARS-CoV-2 RNA and RNase improves molecular detection by preserving SARS-CoV-2 RNA integrity and reveals the prolonged viral loading associated with older age and male gender in COVID-19 patients.

Kandidemi Etkeni Candida albicans Suşlarının Moleküler Yöntemlerle Tiplendirilmesi ve SAP4 Geni Varlığının Araştırılması

Introduction: In recent years, increasing infections due to fungi have drawn attention. Especially, Candida albicans is the most frequent infectious agent with its virulence factors. Furthermore, because C. albicans is a nosocomial infection agent, it can lead an increase in mortality and morbidity. In this study, it was aimed to genotype C. albicans strains that caused candidemia by molecular methods and to investigate one of the most important virulence factors, Secreted Aspartyl Proteinase 4 (SAP4). Materials and Methods: Our study included 50 C. albicans strains isolated from blood cultures. The isolates were identified both phenotypically and genotypically. Arbitrarily-Primed Polymerase Chain Reaction (AP-PCR) and Pulsed Field Gel ElectrophoresisElectrophoretic Karyotype Analysis (PFGE-EK) methods were used for molecular genotyping of the strains. Furthermore, the presence of SAP4 a virulence factor for C. albicans was investigated by polymerase chain reaction (PCR). Results: The 50 C. albicans isolates included in our study were separated to 26 genotypes by AP-PCR, 41 genotypes by PFGE-EK, and SAP4 was detected in 49 of the 50 isolates. Conclusion: In conclusion, dominant genotypes among the C. albicans isolates were not detected. Results of both molecular typing methods revealed that C. albicans isolates were generally endogenous. Furthermore, SAP4 gene was detected in 98% of the isolates. A considerable presence of this gene in blood sample isolates suggests that it contributes to systemic infections. More elaborative studies on the function of SAP4 gene in endogenous infections of C. albicans may contribute to the prevention of these infections in the future.

Antimicrobial Resistance and Molecular Epidemiology of Corynebacterium striatum Isolated in a Tertiary Hospital in Turkey.

Although Corynebacterium striatum is part of the human flora, it has recently drawn attention both for its multidrug resistance and its role as an invasive infection/outbreak agent. This cross-sectional study aimed to determine the antimicrobial resistance and clonal relationships among C. striatum strains. In total, 81 C. striatum strains were identified using Phoenix-100TM (BD, Sparks, MD, USA). The antimicrobial resistance of the strains was determined using the Kirby–Bauer disk diffusion method. Clonal relatedness among the strains was performed via arbitrarily primed polymerase chain reaction (AP-PCR). All 81 C. striatum strains were resistant to penicillin, cefotaxime, ciprofloxacin, and tetracycline, but susceptible to vancomycin and linezolid. The resistance rates to gentamicin, erythromycin, and clindamycin were 34.6%, 79%, and 87.7% respectively. AP-PCR results showed no predominant clone among the C. striatum strains. Corynebacterium striatum is reportedly the cause of an increasing number of invasive infections/outbreaks. Moreover, treatment options are limited. The study showed that vancomycin, linezolid, and gentamicin can be selected for the empirical treatment of C. striatum infections. Although no single-clone outbreak was observed in our hospital, small clonal circulations were observed within some units, indicating cross-contamination. Therefore, a comprehensive infection control program is warranted in future.

Arbitrarily primed polymerase chain reaction-based identification of dystrophic onychomycosis

Background and Objective: Onychomycosis is a fungal infection of the nails; when caused by dermatophytes, it is called as Tinea unguium. The aim was to explicate the utility of arbitrarily primed polymerase chain reaction (AP-PCR) to augment the early and accurate clinical diagnosis of dystrophic onychomycosis. Materials and Methods: The collected nail samples were divided into three portions. The first portion was explored for direct microscopic examination, the second portion was used for culturing of the nail samples on sabouraud dextrose agar media, and the third portion was used for DNA extraction followed by AP-PCR identification of dermatophytes. Results: All 48 samples with dystrophic onychomycosis were diagnosed by three methods, namely, 20% Potassium hydroxide microscopy, culture growth, and AP-PCR. With all the methods, Trichophyton rubrum was found as major causative agents for dermatophyte nail infection (60%), followed by Trichophyton mentagrophytes (23% cases). AP-PCR is a convenient method to achieve a higher dermatophyte identification rate with lesser time and shows complete concordance with conventional culture for two Trichophyton species. Conclusion: AP-PCR is a rapid, specific, and sensitive procedure for the diagnosis of nail fungal infection.

Genotypic variations of Porphyromonas gingivalis in chronic periodontitis patients with and without diabetes: An in vitro study using arbitrarily primed-polymerase chain reaction and heteroduplex-polymerase chain reaction.

Context:Diabetes has become an endemic throughout the world. Periodontitis is associated with diabetes, and it has been regarded as the sixth complication of diabetes. However, the role of diabetes on periodontopathic microbiota such as Porphyromonas gingivalis remains unclear.Aims:To compare the total number of different genotypes of P. gingivalis in chronic periodontitis patients with and without diabetes by using arbitrarily primed-polymerase chain reaction (AP-PCR) and heteroduplex-PCR.Settings and Design:This is a single-center comparative study.Materials and Methods:A total of 100 patients were included in this study. Patients were divided into two groups: Group A – patients having chronic periodontitis without diabetes (50) and Group B – patients having chronic periodontitis with diabetes (50). Subgingival plaque samples were collected from both groups of patients. Plaque samples were cultured for P. gingivalis. Positive culture samples and extracted DNA from samples by proteinase-K method were collected. Part of DNA samples were checked by AP-PCR using OPA-13 primer, and part of the DNA were checked using heteroduplex-PCR using P. gingivalis-specific Pg8 primer. Results were analyzed by polyacrylamide gel electrophoresis procedure.Statistical Analysis Used:Independent t-test and Chi-square test (SPSS 16) were used.Results:Thirty-four out of fifty samples of Group B patients were found positive for P. gingivalis, whereas 21 out of 50 samples in Group A were found positive for P. gingivalis. AP-PCR showed nine different genotypes in Group B and six different genotypes in Group A. Heteroduplex-PCR showed a total of seven different genotypes in Group B and five different genotypes in Group A.Conclusions:Results of this study show that, in diabetic condition, P. gingivalis shows increased variations in genotypes.

Recombinant Methioninase as a DNA Demethylation Agent.

This chapter reviews the effect of methionine (MET) restriction, via treatment with recombinant methioninase (rMETase), on DNA methylation of cancer cells. CCRF-CEM human cancer cells were treated with rMETase under subcytotoxic conditions. The rMETase-treated cells contained significantly lower levels of genomic methylated DNA than did untreated control cells. DNA methylation was measured by incorporation of the methyl group of [3H]methyl-S-adenosylmethionine into DNA and by methylation-sensitive arbitrarily-primed PCR. DNA hypomethylation effected by rMETase was of similar extent to that effected by treatment of the cells with the DNA methyltransferase inhibitor 5-azacytidine.

Horizontal transmission of Streptococcus mutans in children attending kindergarten

Background: Transmission of Streptococcus mutans (S. mutans), the main pathogen found in dental caries, is particularly prevalent during the first two years of infancy. Numerous children are reportedly infected with S. mutans by their mothers with early initial acquisition of the condition considered to carry a higher risk of subsequent dental caries. Purpose: This research aimed to examine the possibility of horizontal transmission of S. mutans in children attending Surabaya-based kindergartens. Methods: The number of subjects who satisfied the inclusion criteria totaled 146. Dental plaque was collected for one minute with a sterile toothbrush. After completion of an isolation process, 25 S. mutans colonies were identified in Tripticase Cysteine Yeast (TYC) media by means of morphological, microscopic and biochemical tests using API 20 Strept (bioMerieux France). A polymerase chain reaction with OPA-2 and 13 was subsequently used to determine the genotype of S. mutans. Primary data collection was completed by the administering of a questionnaire intended to elicit information regarding the gender, age, diet and medical history of subjects. Results: An arbitrarily primed polymerase chain reaction (AP-PCR) fingerprint profile of the strains isolated from the subjects indicated similarities in five genotypes of S. mutans and differences in 18 genotypes. Conclusion: The study indicated that certain children are similarly infected by S mutans bacteria which might be due to horizontal transmission between classmates.

Sensitivity of caries pathogens to antimicrobial peptides related to caries risk.

Antimicrobial peptides (AMPs) represent important facets of the immune system controlling infectious diseases. However, pathogens show varying susceptibilities to AMPs. This study investigates the susceptibilities of strains of Streptococcus mutans (SM), Actinomyces naeslundii (AN), and Lactobacillus spp. (LB) towards AMPs and if there are correlations between the appearance of such high-risk strains and clinical caries status. Plaque samples were collected from patients along with clinical examinations. Bacterial strains were identified via selective media, matrix-assisted laser desorption/ionization analysis-time of flight (MALDI-TOF), and arbitrary-primed-PCR (AP-PCR). Each strain was tested for susceptibility to LL-37, HBD-2, HNP-1, and HNP-3 or phosphate-buffered saline as negative control in a biofilm model on hydroxylapatite discs. Survival rates and resulting risk classification for each strain were determined. Correlations were calculated between the number of high-risk strains (all/S. mutans) appearing in patients and their clinical caries status. Forty-seven patients were included with mean DMFT values of 11.4 ± 8.7. A total of 8 different SM, 30 LB, and 47 AN strains were detected. One-way ANOVA indicated that type/concentration of AMPs had major influence on reductions of Lactobacilli and Actinomyces. Seventeen strains of AN, 2 of SM, and 6 of LB had low susceptibilities to AMPs. The number of such strains in patients showed significant positive correlations to the DMFT values (all p = 0.001; r = 0.452; S. mutans p < 0.0001, r = 0.558). The occurrence of low susceptible strains to AMPs seems to correlate with the individual caries status. The results may lead to new ways to identify individuals with increased caries risk.

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

BackgroundClostridium 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. MethodsWe 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). ResultsSeventeen (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. ConclusionsOur findings suggest that there may be faecal contamination of food products and similar clones of Clostridium perfringens may be incriminated.

Effect of fermented milk containing Lactobacillus rhamnosus SD11 on oral microbiota of healthy volunteers: A randomized clinical trial

The aims of this study were to evaluate whether short-term consumption of fermented milk containing Lactobacillus rhamnosus SD11 affected levels of oral microbiota in vivo and whether L. rhamnosus SD11 could colonize in the human mouth. We also monitored for potential side effects of the probiotic. The applicability of using L. rhamnosus SD11 compared with Lactobacillus bulgaricus as a starter culture for fermented milk was evaluated. After informed consent, 43 healthy young adults were recruited and randomly assigned to either the probiotic or control group and received fermented milk containing L. rhamnosus SD11 or L. bulgaricus, respectively, once daily for 4 wk. The numbers of mutans streptococci, lactobacilli, and total bacteria in saliva were counted at baseline and then after 4 and 8 wk. An oral examination was performed at baseline and after 8 wk. The persistence of L. rhamnosus SD11 was investigated by DNA fingerprinting using arbitrary primer-PCR. Results demonstrated that statistically significant reductions in mutans streptococci and total bacteria were observed in the probiotic group compared with the control group, and the number of lactobacilli was significantly increased in both groups after receiving fermented milks. Lactobacillus rhamnosus SD11 could be detected (in >80% of subjects) up to 4 wk following cessation of dosing among subjects in the probiotic group. No side effects were reported. Thus, L. rhamnosus SD11 could be used as a starter culture for fermented milk. Daily consumption of L. rhamnosus SD11-containing fermented milk for 4 wk may have beneficial effects on oral health by reducing salivary levels of mutans streptococci. The probiotic was apparently able to colonize the oral cavity for a longer time than previously reported. However, the potential benefits of probiotic L. rhamnosus SD11 on oral health require further evaluation with a larger group of volunteers in a longer-term study.