Study on Distribution of Four Pseudomonas Species in Living Environment Using Multiplex PCR

Purpose: The genus Pseudomonas is a ubiquitous microorganism frequently detected from immunocompromised patients. The inherent resistance to numerous antimicrobial agents contributes to the opportunistic character of this pathogen exhaustive monitoring of this pathogen is considered of critical importance to public health organizations. The reliable identification method able to distinguish genetic close Pseudomonas species is needed, because these organisms are difficult to differentiate by phenotypic or biochemical methods. The purpose of the present study was to design species-specific primers in order to identify and detect four Pseudomonas species which are frequently detected from the human oral cavities, and to investigate the distribution of these organisms in the living environment using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the rpoD gene of four Pseudomonas species. Swab samples were collected from fifty washstands, and the distribution of Pseudomonas species was investigated using a conventional PCR at genus level and a multiplex PCR at species level. Results: Multiplex PCR method developed in this study was able to distinguish four Pseudomonas species clearly. The genus Pseudomonas was detected from all samples (100%), whereas P. putida, P, aeruginosa, P. stutzeri and P. fluorescens were detected at 44%, 8%, 4% and 2% in fifty swab samples, respectively. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and works without requiring DNA extraction. It was indicated that washstands were the uninhabitable environment for P. putida, P, aeruginosa, P. stutzeri and P. fluorescens.

Investigation of Bacteria Species Most Involved in Peri-Implantitis

Purpose: Currently, bacteriological examinations of implant treatments target periodontopathic bacteria such as red complex bacteria, including Porphyromonas gingivalis, and detect them qualitatively or quantitatively. However, it seems that those examinations do not reflect the peri-implant tissue conditions precisely, because periodontopathic bacteria are also frequently detected from healthy peri-implant sites. The purpose of the present study was to investigate bacteria species most involved in peri-implantitis using a PCR method. Methods: Polymerase chain reaction (PCR) primers in this study were designed based on partial sequences of 16S rDNA of bacteria species involved in peri-implantitis that were described in numerous previous studies. Peri-implant sulcus fluid (PISF) samples were collected from thirty periodontally healthy patients with implants (HI) and thirty patients with peri-implantitis (PI). Each detection frequency of bacteria species in PISFs of both groups was investigated using a PCR method, and was compared using Fisher’s exact test. Results: In PI group, detection frequencies of Corynebacterium durum, Fretibacterium fastidiosum and Slackia exigua were significantly higher than those of HI group (p P. gingivalis and Tannerella forsythia belonging to red complex were frequently detected in the PISF samples of HI group (p > 0.05). Conclusion: It was suggested that monitoring C. durum and F. fastidiosum levels in PISF samples was useful as a clinical indicator for the evaluation of peri-implant tissue conditions.

Isolation and Identification Methods for Oral Klebsiella pneumoniae Involved in Onset of Inflammatory Bowel Disease

Purpose: Recently, it was reported that Klebsiella pneumoniae is related to the onset of inflammatory bowel disease including the Crohn disease. It was frequently reported that K. pneumoniae was detected in human oral cavities. Regrettably, it currently remains unclear whether K. pneumoniae is part of the normal oral flora. The aim of this study was to establish the isolation and identification methods for K. pneumoniae from human oral cavities, and investigate its transmission pattern. Methods: A selective medium, OKPSM, for the isolation of K. pneumoniae from oral cavities was developed in this study. Also, PCR primer for the identification and detection at subspecies level of K. pneumoniae was designed. Results: OKPSM and PCR method using the primers designed in this study were useful for the isolation and identification of K. pneumoniae from human oral cavities. K. pneumoniae subsp. pneumoniae was detected at 10.0% in 30 saliva samples. On the other hand, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis were detected from no sample. Moreover, K. pneumoniae subsp. pneumoniae isolates from same subject at 0 month and after 3 months showed same genotypes on AP-PCR using OPA-07 primer. Conclusion: These results indicated that human oral cavities were not suitable for the habitat of K. pneumoniae.

Study on Distribution of Acinetobacter baumannii Complex in Dental Hospital Using Multiplex PCR

Purpose: In recent years, multidrug-resistant Acinetobacter baumannii has appeared and caused outbreaks of hospital infections all over the world. Close monitoring of this pathogen and other A. baumanii complex species is considered of critical importance to public health organizations. The reliable identification method able to distinguish A. baumannii from genetically close Acinetobacter species is needed, because these species are unable to be differentiated by phenotypic or biochemical methods. The purpose of the present study was to design species-specific primers in order to identify and detect A. baumanii complex species, and Acinetobacter lwoffii which is frequently detected from the human specimens, and to investigate the distribution of these organisms in dental hospital using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 16S ribosomal RNA (16S rRNA) gene and DNA gyrase subunit B (gyrB) of each species of A. baumanii complex species. Swab samples were collected from ten dental spittoon units in dental hospital, and the distribution of A. baumanii complex species was investigated using a multiplex PCR. Results: These primers were able to distinguish each species of A. baumanii complex species clearly. A. baumanii and A. calcoaceticus were detected at 20.0% and 10.0% in ten swab samples, respectively. On the other hand, A. nosocomialis, A. lowffii, and A. pittii were detected from no sample. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.

One Step Multiplex PCR for Identification of Candida haemulonii complex and Candia auris

Purpose: Recently, Candida haemulonii complex (Candida haemulonii, Candida duobushaemulonii and Candida haemulonii var. vulnera) and two genetically close species (Candida pseudohaemulonii and Candida auris) have emerged as an opportunistic fungal pathogen associated with various infectious diseases of humans, and most of those isolates have displayed antifungal resistance. Because it is difficult to differentiate these microorganisms by a current technique, unfortunately, it is important to establish a method for identifying them accurately. The purpose of the present study was to design species-specific primers in order to identify and detect C. auris, C. pseudohaemulonii, and C. haemulonii complex, i.e. , C. haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 26S rRNA, 18S rRNA, and RPB1 genes and ITS region of five Candida species. Results: The multiplex PCR method developed in this study was able to distinguish five Candida species clearly. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and works without requiring DNA extraction.

Study on the Distribution at Species Level of Genus Candida in Human Oral Cavities, Using Culture and Multiplex PCR Methods

Purpose: Although the genus Candida is frequently isolated from human oral cavities, the distribution at the species level of these organisms has been little reported. The purpose of the present study was to assess the distribution at the species level of the genus Candida in human oral cavities. Methods: This study was performed using culture and Multiplex PCR methods. Moreover, the genotyping classification of C. albicans was analyzed with a PCR. Results: Of all subjects (n = 90), detection frequency of genus Candida was 42.2%. Genus Candida was not detected in the subjects between 0 to 9 years old, and there was no difference in the detection frequencies of this organism among each generation from 10s to 80s. C. albicans was the most dominant species, followed by C. parapsilosis, C. glabrata, and C. dubliniensis. Plural Candida species tended not to be detected in the individual sample. Genotype A was dominant in the C. albicans isolates. Conclusion: These results indicated that C. albicans of genotype A was dominant and that the genus Candida rarely coexists with other Candida species, in each individual oral cavity.

Origin of Candida albicans 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.

One Step Multiplex PCR for Identifications at Subspecies Level of Fusobacterium nucleatum and Fusobacterium necrophorum

Purpose: Fusobacterium nucleatum is an opportunistic pathogen involved in periodontal diseases, extraoral infections, and colorectal cancer. Fusobacterium necrophorum causes a variety of necrotic infections. F. nucleatum and F. necrophorum are classified into five and two subspecies, respectively. Conventional identification methods were technically hard to distinguish each subspecies of two Fusobacterium species accurately. The purpose of the present study was to design primers to identify two medically important Fusobacterium species at the subspecies level, using one-step multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 16S ribosomal RNA (16S rRNA) gene, RNA polymerase B (rpoB) gene, and DNA gyrase subunit B (gyrB) of each subspecies of F. nucleatum and F. necrophorum. Results: These primers were able to distinguish each subspecies of F. nucleatum and F. necrophorum and did not display cross-reactivity with representative Fusobacterium species other than F. nucleatum and F. necrophorum. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.

Isolation and Identification Methods for Actinomyces israelii Involved in Actinomycosis

Purpose: Actinomyces israelii is known as the key species to cause classical actinomycosis. Although A. israelii is frequently isolated from human oral cavities, the distribution of this microorganism has been little reported. The purpose of the present study was to develop selective media (AISM) for the isolation of A. israelii and to assess the prevalence of this organism in the oral cavity. Methods: To examine the bacterial population in the oral cavity, a novel selective medium (AISM) was developed for isolating A. israelii. AISM consists of BHI, yeast extract, agar, ofloxacin, fosfomycin, colistin, and sodium fluoride. Results: A. israelii strains grew well on AISM. A. israelii was detected in all dental plaque samples collected from 20 subjects and the mean number of this organism in the samples was 7.9 × 104 CFU/ml. Conclusion: These results indicated that the selective medium was useful for the isolation of A. israelii and this organism was a part of the normal flora in the human oral cavity.