Efficiency of Various Growth Media in Recovering Oral Bacterial Flora from Human Dental Plaque

MM10 sucrose blood agar (MM10 SB agar), N(2)C agar, Schaedler agar (SH agar), and mitis salivarius agar (MS agar) were tested for their ability to recover human dental plaque flora by a continuous anaerobic procedure and by a conventional anaerobic method. MM10 SB agar yielded higher recovery of bacteria from plaque samples as determined by the enumeration of colony-forming units (CFU). The CFU on N(2)C agar, SH agar, and MS agar were lower than MM10 SB agar when the continuous anaerobic procedure was used. The superior performance of MM10 SB agar was much more apparent when used for the cultivation of dental plaque by the conventional anaerobic method. Under these conditions the counts were consistently higher on MM10 SB agar as compared to the other media tested. However, the differential counts of Streptococcus sanguis and S. mutans from carious plaque samples were in general comparable on all culture media. Deletion of blood from MM10 SB agar did not lower counts. The elimination of dithiothreitol from this medium resulted in a significantly lower recovery of bacteria from the plaque samples when cultured by the conventional anaerobic method. The storage of MM10 SB agar for varying periods of time aerobic conditions did not seem to affect its performance. These findings suggest that MM10 SB agar is an ideal culture medium for the isolation, nonselective enumeration, and differential counts of bacteria present in normal and disease-associated plaques.     

固定矫治器戴入前后牙周可疑病原菌和牙周临床指数的变化

目的:固定矫治器戴入前后的不同时间进行龈缘菌斑的微生物学检查和牙周状况的临床检查,以探讨固定矫治器戴入前后牙周可疑病原菌和牙周状况的动态变化过程.方法:选择18名固定正畸患者,于矫治器戴入前和戴入后1、3、6月分别检查16、41牙位的菌斑指数、牙龈指数、探诊深度、探诊出血,并在近中颊侧颊轴角处采集龈缘菌斑样本,采用细菌培养鉴定方法测定牙周可疑病原菌的检出量(CFU/g)和检出率.结果:与基线相比,观察期内龈缘菌斑的G￣产黑色素厌氧杆菌检出量和检出率在两个牙位均升高(P＜0.05),41的优杆菌、弯曲杆菌、拟杆菌、普氏菌亦有升高(P＜0.05).临床指标中16的牙龈指数(颊侧)和探诊深度(颊、舌侧)升高;41的菌斑指数(舌侧)降低(P＜0.05).结论:固定矫治器戴入后虽然可通过严格的口腔卫生指导有效控制牙面菌斑,但仍可引起牙周可疑病原菌增加.     

Porphyromonas gingivalis prevalence related to other micro-organisms in adult refractory periodontitis.

Forty-six adult periodontal patients, selected on the basis of clinical examination, and 46 adult healthy subjects were examined. The subgingival plaque samples from one inflammatory and one non-inflammatory site of each periodontal patient were studied to determine Porphyromonas gingivalis prevalence related to other periodontal micro-organisms and to periodontal tissue destruction. The results showed Porphyromonas gingivalis as the main pathogenic micro-organism isolated in the inflammatory sites together with Bacteroides forsythus. Peptostreptococcus sp., Actinomyces sp. and Prevotella sp. were found as a normal oral flora in the healthy subjects. Fusobacterium nucleatum, Prevotella intermedia, Campylobacter rectus and Eikenella corrodens were detected both in inflammatory and in non-inflammatory sites of periodontal patients as well as in the healthy subjects.     

Transport Media for Herpes Simplex Virus Types 1 and 2

An evaluation was made of the recovery rate of herpes simplex virus (HSV) type 1 or 2 from 197 clinical specimens obtained in two or three charcoal transport media: Leibovitz viral transport medium, a modified Leibovitz-Emory medium (LEM), in which agarose was used instead of agar, and Amies bacterial transport medium. The specimens were stored and shipped for 1 to 19 days in these media at ambient temperature or in Hanks buffered-salt solution in dry ice. The results indicate that the LEM was most effective, particularly in the recovery of HSV type 2 from clinical specimens held at ambient temperature. In vitro and in vivo studies in genitally infected mice corroborated the observations obtained with human clinical specimens. The availability of transport media which can be used for shipment at ambient temperature offers clinicians easier accessibility to laboratory confirmation and antigenic typing of HSV from suspect herpetic infections.     

Effect of the Conditions of Incubation in the Recovery of Oral Actinomyces

The Gram-positive Pleomorphic Bacilli (GPPB), especially the genus Actinomyces, are part of the oral microflora and they are generally associated with cement caries. They are also found in inactive sites in periodontal disease and in pulpar infections. The aim of the present work was to find an appropriate isolation culture medium for the recovery of the genus Actinomyces from oral samples, and to propose a minimum schema of biochemical tests for the identification and differentiation of Actinomyces species from other GPPB. Samples of saliva, subgingival plaque and post-extraction acute alveolar osteitis were cultivated in Starch Casein Agar (SCA), Yeast Extract Dextrose Agar (YDA), Columbia Blood Agar 5% (BA), both with cephadroxyl (10 μL/mL) and without antibiotic. The plates were incubated in aerobic conditions, in strict anaerobic conditions and in a candle jar for 5 days at 37°C. (1) The highest recovery of Actinomyces was obtained in BA without antibiotic in a candle jar for all oral samples. (2) In all the incubated in SCA, YDA and BA with antibiotic,Actinomyces , other GPPB, Gram-positive and -negative cocci and Gram-negative bacilli showed growth in all aerobic conditions. (3) In BA medium with antibiotic the Gram-negative microflora was inhibited, the Actinomyces recovery being lower than in the other media studied.     

Recovery of Anaerobic Microorganisms from Clinical Specimens in Prereduced Media Versus Recovery by Routine Clinical Laboratory Methods

Prereduced anaerobically sterilized culture media, used with rigid adherence to the cultivation techniques described by Moore and his associates, were capable of recovering more than twice the number of anaerobic bacteria from clinical specimens than could be recovered by the conventional use of fluid thioglycolate medium and of blood-agar plates incubated anaerobically with hydrogen generation packets. No loss of clinical isolates was encountered with the more sensitive methods; however many of the isolates recovered only in prereduced media would not grow when placed into thioglycolate medium. A representative anaerobic isolate placed into aerobic transport broth was unable to survive beyond 30 min. Methods employing prereduced media were not difficult to master and were feasible for clinical laboratory use. Evidence implicating the gingival crevice flora as an important possible source of anaerobic bacteria that become involved in systemic infections was considered.     

Analysis of the oral bacterial flora in subjects with periodontal diseases

A bacterioscopic study of the bacterial flora of 15 healthy and 15 periodontal disease carriers is presented in relation to the possibly pathogenetic role of oral bacterial flora in the development of human periodontal disease. Study samples taken from sulcus and gingival pockets were examined by darkfield microscopy. Results show that bacterial flora from periodontal disease subjects differs from that in healthy subjects in that spirochetes are present and the incidence of straight and motile rods is higher than of coccoid cells.     

Physiological Aspects and Conservation of a Veillonella Strain Isolated From the Oral Cavity. Interaction with Streptococci

Bacteria of the genus Veillonella are anaerobic, Gram-negative cocci which are found as normal flora in the human oral cavity. Because it grows well with the lactate produced by streptococci, antinomyces and lactobacilli in dental plaque, they could play an anticariogenic role. The aim of the present study was to investigate the kinetics and viability of a Veillonella strain isolated from saliva and its interaction with oral streptococci. The growth rate was determined in Lactate broth measuring turbidity and CFU/mL over 64 h. Preservation media were tested at −70°C, −20°C and room temperature to strain conservation. To study bacterial interactions between veillonella and streptococci, an active culture of veillonella was spread on Mitis Salivarius Agar plates, which is a culture medium selective for oral streptococci. The replication time of this veillonella strain was 7.2 h in Lactate Broth and the specific growth rate (μ) was 0.096 h. The elected medium for conservation was Preservation Broth with 25% Glycerol at all temperatures. The Muñiz Maintenance Medium and Muñiz Maintenance Medium with Glycerol 25% media may be used at any temperature for short time periods. The interaction between veillonella and streptococci seems to be a result of nutritional factors.     

Presence of Candida albicans in the oral cavity of healthy subjects

The influence of possible changes in bacterial flora in the etiology of periodontal disease assessed during a study of saprophitism of Candida albicans in the oral cavity is reported. Three saliva samples, each from each of 50 healthy subjects not on drugs, taken prior to breakfast and after careful cleaning of the oral cavity the previous evening were subjected to bacterioscopic examination both immediately and after culturing on Sabouraud medium. pH levels were also determined. 17 subjects, of which females predominated, (12 cases compared to 5 male cases) were found to be C.a carriers.     

液相BANA试验检测口腔关键厌氧菌方法的建立

目的建立液相苯甲酰精氨酸萘酰胺(简写BANA)水解试验快速检测牙周病关键厌氧菌的新技术。方法利用牙周病相关关键微生物可产生胰蛋白酶样酶使人工合成的酶底物———BANA水解的原理检测这些重要口腔厌氧菌。结果BANA试验可检测出8.0×105以上的牙龈卟啉菌,特异性良好、不受标本采集中血污染的影响;71.88%活动期牙周炎龈下菌斑BANA试验阳性,4%的健康人龈下菌斑BANA试验阳性。结论BANA试验可望成为牙周病诊断新的辅助指标。     

The Effects of Nisin on the Microbial Flora of the Dental Plaque of Monkeys (Macaca fascicularis)

Nisin was incorporated in a soft diet and fed to eight monkeys for five months. At the end of this period dental plaque was removed from one site in the mouths of these animals and from a similar site in eight control animals. Twelve different selective media were used for the cultivation of the bacteria in the plaque. Streptococci, Haemophilus spp. and Gram negative anaerobes were the organisms most frequently isolated. There was some evidence that streptococci constituted a lesser proportion of the flora in the nisin-fed animals.     

健康青少年口腔正常菌群的初步研究

本文采用非选择性培养基对22名健康青少年的唾液、沟裂菌斑、龈上菌斑及龈下菌斑中的需氧菌、兼性厌氧菌及专性厌氧菌进行了分离培养,并计算其在不同标本中占可培养菌的百分比及检出率。结果共分离到包括18个菌属的35种细菌。其中,链球菌、放线菌、奈瑟氏球菌、二氧化碳噬纤维菌、类杆菌、梭杆菌,奴卡氏菌及棒状杆菌在口腔4个部位的检出率及所占比例均较高,是健康青少年口腔中的优势菌群.通过比较还发现,其中一些菌在口腔4个部位的分布存在一定差异.本文还采用刚果红负性染色涂片法,镜下观察龈上、龈下菌斑中的螺旋体,并计算其相对比例.结果龈下菌斑中螺旋体的相对比例明显高于龈上菌斑.     

Selectivity of Mitis Salivarius agar and a new selective medium for oral streptococci in dogs

An evaluation on the applicability of Mitis Salivarius agar (MS) medium, commonly used for the detection of oral streptococci in human and animals, to dog specimens and the development of a new selective medium for isolating streptococci from the canine oral cavity are described. Oral samples from dogs were cultured on MS medium under anaerobic conditions. The predominant facultative anaerobic bacteria on MS plates were gram-negative rods. Selectivity of streptococci on MS medium was 21.2%. A new selective medium, designated MS-CAN-AE, was developed for the isolation of streptococci from the canine oral cavity. The average growth recovery of laboratory and clinically isolated strains of streptococci on MS-CAN-AE medium was 84.1% of that on MS medium. Gram-positive rods and gram-negative rods and cocci rarely grew on the MS-CAN-AE. The selectivity of MS-CAN-AE was 95.0% for clinical samples. MS-CAN-AE medium will be helpful for investigations of streptococci in the canine oral cavity.     

Quantitative Molecular Detection of Putative Periodontal Pathogens in Clinically Healthy and Periodontally Diseased Subjects

Periodontitis is a multi-microbial oral infection with high prevalence among adults. Putative oral pathogens are commonly found in periodontally diseased individuals. However, these organisms can be also detected in the oral cavity of healthy subjects. This leads to the hypothesis, that alterations in the proportion of these organisms relative to the total amount of oral microorganisms, namely their abundance, rather than their simple presence might be important in the transition from health to disease. Therefore, we developed a quantitative molecular method to determine the abundance of various oral microorganisms and the portion of bacterial and archaeal nucleic acid relative to the total nucleic acid extracted from individual samples. We applied quantitative real-time PCRs targeting single-copy genes of periodontal bacteria and 16S-rRNA genes of Bacteria and Archaea. Testing tongue scrapings of 88 matched pairs of periodontally diseased and healthy subjects revealed a significantly higher abundance of P. gingivalis and a higher total bacterial abundance in diseased subjects. In fully adjusted models the risk of being periodontally diseased was significantly higher in subjects with high P. gingivalis and total bacterial abundance. Interestingly, we found that moderate abundances of A. actinomycetemcomitans were associated with reduced risk for periodontal disease compared to subjects with low abundances, whereas for high abundances, this protective effect leveled off. Moderate archaeal abundances were health associated compared to subjects with low abundances. In conclusion, our methodological approach unraveled associations of the oral flora with periodontal disease, which would have gone undetected if only qualitative data had been determined.     

Oxygen metabolism,oxidative stress and acid-base physiology of dental plaque biofilms

Dental plaque is a natural biofilm which has been a focus of attention for many years because of its known roles in caries and periodontal diseases. Acid production by plaque bacteria leads to the erosion of tooth mineral in caries, and the cariogenicity of plaque is related to population levels of acid-tolerant organisms such as mutants streptococci. However, the biofilm character of plaque allows for survival of a diverse flora, including less acid-tolerant organisms, some of which can produce ammonia from arginine or urea to counter acidification. Plaque is often considered to be relatively anaerobic. However, evidence is presented here that both supragingival and subgingival plaque have active oxygen metabolism and that plaque bacteria, including anaerobes, have developed defenses against oxidative stress. Even in subgingival plaque associated with periodontitis, measured residual oxygen levels are sufficient to allow for oxygen metabolism by organisms considered to be extremely anaerobic such asTreponema denticola, which metabolizes oxygen by means of NADH oxidases and produces the protective enzymes superoxide dismutase and NADH peroxidase. The finding that plaque bacteria produce a variety of protective enzymes is a good indicator that oxidative stress is a part of their everyday life. The biofilm character of plaque allows for population diversity and coexistence of aerobes, anaerobes and microaerophiles. Overall, agents that affect oxidative metabolism offer possibilities for reducing the pathogenic activities of plaque.     

Bronchopneumonia and oral health in hospitalized older patients. A pilot study

Aims: To correlate microbial findings obtained by bronchoalveolar lavage in pneumonia patients with the clinical situation of the oral cavity. Method: Quantitative aerobic and anaerobic cultures were carried out in 150 ml samples of bronchoalveolar lavage (BAL) obtained by means of an endoscope (Video Endoscope Pentax®) inserted per as in the infected bronchus. Material: Twenty consecutive patients with a tentative clinical diagnosis of bronchopneumonia in whom BAL was carried out for diagnostic purposes. A clinical evaluation of the oral health status (oral hygiene, caries, periodontal diseases) was subsequently carried out. Results: In seven edentulous subjects wearing complete dentures the culture of anaerobic microorganisms was negative or yielding less than 100 cfu/ml BAL. Two patients yielded high counts of S. aureus and one high counts of P. aeruginosa. In the 13 subjects with natural teeth left one showed high counts of Veillonella spp. (anaerobic)+P. aeruginosa, one high counts of Veillonella spp. +S. aureus, one high counts of P. aeruginosa + S. aureus and one high counts of E. coli. These four subjects showed poor oral hygiene, periodontal pockets and a BAL microflora consistent with periodontal pathology. Conclusion: The results of this pilot study suggest that microorganisms of denture plaque or associated with periodontal diseases may give rise to aspiration pneumonia in susceptible individuals.     

Comparison of Methods for Isolation of Anaerobic Bacteria from Clinical Specimens

Five different anaerobic culture methods and several different media were compared for their ability to recover anaerobes from clinical specimens. Specimens were obtained from patients with documented infections, avoiding contamination with normal flora, and immediately placed in an anaerobic transporter. Each specimen was cultured by all methods and on all the various media. The comparative data indicate that anaerobic jars (GasPak and evacuation-replacement types) are just as effective in the recovery of clinically significant anaerobes as the more complex roll-tube and chamber methods employing prereduced media. Liquid media were disappointing as a "back-up" system but chopped-meat glucose was superior to two thioglycolate formulations. Growth of all anaerobes was poorer on selective media, but these media were very helpful in the workup of specimens containing mixed growth of anaerobic and facultative organisms. A variety of different anaerobes was isolated, but no very fastidious or extremely oxygen-sensitive organisms were recovered. This suggests that such organisms may not play a significant role in causing clinical infections.     

Coaggregation properties of human oral Veillonella spp.: relationship to colonization site and oral ecology.

The primary habitats of oral veillonellae are the tongue, dental plaque, and the buccal mucosa. Isolates were obtained from each habitat and tested for coaggregation with a battery of other oral bacterial strains. All 59 tongue isolates tested for coaggregation were Veillonella atypica or Veillonella dispar. All but one of them coaggregated with strains of Streptococcus salivarius, a predominant inhabitant of the tongue surface but not subgingival dental plaque. These tongue isolates were unable to coaggregate with most normal members of the subgingival flora such as Actinomyces viscosus, Actinomyces naeslundii, Actinomyces israelii, and Streptococcus sanguis. In contrast, 24 of 29 Veillonella isolates, of which 20 were Veillonella parvula from subgingival dental plaque samples, coaggregated strongly with the three species of Actinomyces, S. sanguis, and other bacteria usually present in subgingival plaque, but they did not coaggregate with S. salivarius. The majority of isolates from the buccal mucosa (42 of 55) has coaggregation properties like those from the tongue. These results indicate that the three human oral Veillonella species are distributed on oral surfaces that are also occupied by their coaggregation partners and thus provide strong evidence that coaggregation plays a critical role in the bacterial ecology of the oral cavity.     

Coaggregation properties of human oral Veillonella spp.: relationship to colonization site and oral ecology

The primary habitats of oral veillonellae are the tongue, dental plaque, and the buccal mucosa. Isolates were obtained from each habitat and tested for coaggregation with a battery of other oral bacterial strains. All 59 tongue isolates tested for coaggregation were Veillonella atypica or Veillonella dispar. All but one of them coaggregated with strains of Streptococcus salivarius, a predominant inhabitant of the tongue surface but not subgingival dental plaque. These tongue isolates were unable to coaggregate with most normal members of the subgingival flora such as Actinomyces viscosus, Actinomyces naeslundii, Actinomyces israelii, and Streptococcus sanguis. In contrast, 24 of 29 Veillonella isolates, of which 20 were Veillonella parvula from subgingival dental plaque samples, coaggregated strongly with the three species of Actinomyces, S. sanguis, and other bacteria usually present in subgingival plaque, but they did not coaggregate with S. salivarius. The majority of isolates from the buccal mucosa (42 of 55) has coaggregation properties like those from the tongue. These results indicate that the three human oral Veillonella species are distributed on oral surfaces that are also occupied by their coaggregation partners and thus provide strong evidence that coaggregation plays a critical role in the bacterial ecology of the oral cavity.     

Effects of oxygen on the growth and metabolism of Actinomyces viscosus

Abstract Actinomyces viscosus is a predominant microorganism in dental plaque. It is, just as the oral Streptococcus spp., a saccharolytic and aero-tolerant organism. We have investigated the effects of oxygen on the growth and metabolism of A. viscosus . To this end A. viscosus Ut 2 was grown in a glucose limited chemostat culture on a chemically defined medium ( D = 0.2 h⁻¹) with exposure to variable amounts of oxygen. The Y_glucose increased from 62.5 g · mol⁻¹ under anaerobic conditions to 149 g · mol⁻¹ under aerobic conditions, while, concomitantly, the carbon recovery from acidic fermentation products decreased from 75% to 7%. Addition of [¹⁴C]glucose to the chemostat showed that the glucose, which was not converted to acidic fermentation products, was instead converted to carbon dioxide or used for the production of biomass. Under aerobic and anaerobic conditions identical cytochrome spectra, containing only two cytochrome b -type absorption bands, were found. It was concluded that electron transport phosphorylation probably occurs both under aerobic and anaerobic conditions. Anaerobically, fumarate served as the electron acceptor, while the high growth yields observed under aerobic conditions are likely to be explained by citric acid cycle activity coupled to electron transport phosphorylation.