Genotypic diversity of Streptococcus mutans in 3- to 4-year-old Chinese nursery children suggests horizontal transmission

OBJECTIVE: The aims of this study were to investigate the colonisation of Streptococcus mutans and to determine the possibility of horizontal transmission in Chinese nursery children. DESIGN: Fifty-six children aged between 3 and 4 years old from a nursery were included in this study. Twenty-four children were from the day and night nursery class, the others were from the day nursery class. Dental plaque samples were collected with sterile toothpicks and cultured on S. mutans-selective tryptone-yeast-cystine agar supplemented with 0.2U/ml bacitracin and 15% sucrose. The typical isolates of S. mutans were identified by classical microbiological methods and genotyped by arbitrarily primed PCR (AP-PCR) and restriction fragment length polymorphism analysis. RESULTS: S. mutans was isolated from 41 of the 56 children. The prevalence of S. mutans was higher in the children from day nursery class, compared with the children from day and night nursery class (P<0.05). A total of 140 S. mutans isolates from 41 children were analysed by AP-PCR, and 88 different amplitypes were identified. Of 41 children with S. mutans isolates, 82.9% carried two or more genotypes. Two genotypes of S. mutans were repeatedly isolated among 13 children in the day and night nursery class, and one genotype was isolated from two children in the day nursery class. CONCLUSION: The presence of matching genotypes of S. mutans among children attending the same nursery suggests the possibility of horizontal transmission.     

PCR with arbitrary primers: approach with care

New techniques have recently been described that employ the polymerase chain reaction (PCR) to amplify arbitrary regions of a genome using a single primer. The techniques reveal polymorphisms in insect taxa that lack allozyme variation and, for the first time, permit genetic polymorphisms to be rapidly analysed in small arthropods (e.g. mites, endoparasitic wasps). The methods have been used in identification of sub-species and cryptic species, and have applications in population genetics and genetic fingerprinting. They are fairly inexpensive, do not require the use of radioactivity, are relatively simple to learn and can easily be adapted to most laboratories. However, their application is not without technical problems and practical limitations. The purpose of this note is to indicate the critical factors to consider before launching into their use. We chiefly emphasize that most polymorphisms revealed by these methods segregate as dominant markers. Furthermore, application of these techniques requires extensive standardization and may not prove to be reproducible among various laboratories especially those employing different types of thermal cyclers. There are some unique features of these polymorphisms to consider when using them in genetic fingerprinting. In addition, because the techniques amplify arbitrary regions of genomes, similarly sized fragments amplified between two species may not be homologous. This argument and empirical observations suggest that PCR with arbitrary primers will have limited application in molecular systematics above the intraspecific level.     

Survey of methicillin-resistant coagulase-negative staphylococci isolated from the fingers of nursing students

 To clarify the state of methicillin-resistant coagulase-negative staphylococci (MRCNS) contamination in the hospital environment, we compared MRCNS isolated from the fingers of 40 nursing students who had not yet experienced clinical practice and 40 who had just completed clinical practice in the hospital. Fourteen MRCNS strains were detected in 13 students (32.5%) after clinical practice; Staphylococcus epidermidis in 9 students, Staphylococcus haemolyticus in 3, and Staphylococcus saprophyticus in 2. Drug sensitivity tests were performed, and the minimum inhibitory concentration (MIC) of penicillin-G (PCG) was more than 2 μg/ml in all strains, and that of ampicillin (ABPC) was more than 16 μg/ml in many strains. Only a few strains showed high MIC values for the other drugs tested. However, some Staphylococcus haemolyticus strains showed high MIC values for cefazolin (CEZ), arbekacin (ABK), gentamicin (GM), ofloxacin (OFLX), or imipenem (IPM). In all strains, the mecA gene was detected by polymerase chain reaction (PCR), and penicillin binding protein 2′ (PBP2′) was detected by the latex agglutination method. Methicillin-resistant Staphylococcus epidermidis (MRSE) isolated from the fingers of nursing students was compared with that isolated from blood culture specimens by arbitrarily primed (AP)-PCR analysis. The patterns obtained were different, a finding which excluded the presence of cross-infection. The present results show that basic preventive measures for cross-infection should be considered in the future, using such genetic analysis methods, so that MRCNS may not cause hospital infection. Received: May 27, 2002 / Accepted: September 9, 2002     

江苏省M_1型伤寒沙门氏菌的分子流行病学特征

选择江苏省各地 1988～ 1997年分离到的 2 6株M1型伤寒菌进行AP -PCR分析。结果 2 6株M1型伤寒菌被引物 2 2 732、2 2 733和 2 2 734共同划分为 7种不同的基因型别。将菌株的基因型别与流行病学资料分析相结合 ,发现在江苏省伤寒呈现高强度流行的 80年代末至 90年代初 ,M1型菌株呈现高度的基因多态性表现 ,而在江苏省伤寒流行强度较低的近几年 ,M1型菌株的基因多态性表现则明显减弱。     

Genomic alteration is not associated with fatty and clear cell change in hepatocellular carcinomas and its precursor nodular lesions in cirrhotic liver.

BACKGROUND: The aim of this study was to investigate whether fatty and clear cell areas in large regenerative nodules (LRN), dysplastic nodules (DN), and hepatocellular carcinoma (HCC) show higher degree of genomic mutation compared to non-fatty/clear cell area in the same nodule or non-lesional tissue. METHODS: We examined 22 nodular lesions (9 HCC, 5 DN and 8 LRN) from seven cirrhotic livers removed at transplantation. Frozen sections were used for manual microdissection of areas with fatty/clear cell change. DNA from microdissected tissue was amplified using arbitrarily primed polymerase chain reaction (AP-PCR), and PCR products were run on polyacrilamide gel generating a "fingerprint" band pattern. Autoradiographs were analysed using Adobe Photoshop version 6.0. Fingerprints from lesional tissue were compared to reference tissue and the total number of bands in excess or defect was calculated and divided by the total number of bands identified, obtaining the genomic damage fraction (GDF). RESULTS: Increasing GDF average values were seen from cirrhotic liver (0.13+/-0.04), to LRN (0.16+/-0.1), DN (0.28+/-0.08) and HCC (0.30+/-0.07). A statistically significant difference in GDF values was documented between cirrhotic liver and DN (p=0.008) and HCC (p=0.005) and between HCC and LRN (p=0.02). No significant difference was documented between DN and HCC, and between LRN and cirrhotic liver. Eleven nodules containing fat/clear cell areas were compared to the other 11 nodules without fat/clear cell areas. The GDF was not different between the two groups: 0.29+/-0.11 versus 0.25+/-0.12; p=0.5. The average value of genomic damage fraction between fat/clear cell areas (0.29+/-0.11) and no fat/clear cell areas (0.25+/-0.1) within the same nodules were not significantly different (p=0.11). CONCLUSION: Fatty and clear cell change in nodular lesions in cirrhotic liver may be an epigenetic phenotypic modification caused by microenvironmental factors such as ischaemia rather than indicating areas of increased malignant potential per se.     

临床常见皮肤癣菌的分子生物学鉴定

目的应用任意引物聚合酶链反应(AP-PCR)鉴定临床常见皮肤癣菌。方法用引物AP3(5-′TCAC-GATGCA-3′)、任意引物ATG(5-′ATGGATCGGC-3′)、任意引物TCA(5-′TCACCACGGT-3′)和任意引物TCT(5-′TCTGTGCT-GG-3′)对常见皮肤癣菌进行任意引物聚合酶链反应。46株须癣毛癣菌采用引物ATG和引物AP3进行AP-PCR,并对扩增产物进行电泳分析。结果扩增产物电泳分析发现不同菌种间产生的DNA带型存在明显差异,尤其用引物AP3和引物ATG扩增产生的DNA带型的种间差异最明显。46株须癣毛癣菌的表型,全部为粉型,且均产生具有明显的电泳带型,可分为2型。结论AP-PCR可作为常见皮肤癣菌的种间及种内鉴定的简单、快速、可靠的方法。     

随意引物聚合酶链反应在健康青少年口腔优势菌鉴定中的应用

目的：应用随意引物聚合酶链反应(arbitrarily primed polymerase chain reaction，AP-PCR)来对健康青少年口腔优势菌进行鉴定。方法：经初步鉴定健康青少年口腔优势菌为血链球菌属后，用随机引物AP-PCR对优势菌进行扩增、检测。结果：经扩增后的健康青少年口腔优势菌的DNA片段与标准血链球菌ATCC10556的DNA片段相同。结论：应用AP-PCR技术可以对健康青少年口腔优势菌进行鉴定和分类。     

布鲁氏菌AP-PCR最优反应体系的建立

目的建立布鲁氏菌的随机扩增DNA多态性的优化反应体系，用于布鲁氏菌分子流行病学研究。方法利用几种随机引物及其组合(Pl、P2、P3、P4、P5、OPLO4、P3／5、P4／5)进行随机引物PCR的反应(AP—PCR，又称RAPD)，及重复片断引物ERICIR／ERIC2的随机扩增多态性；另外针对优选引物P5和OPLO4，分别对Mg^2 浓度、dNTPs浓度、模板DNA浓度、引物的浓度及扩增过程中的退火温度等影响反应结果的因素，进行了一系列优化组合方案的试验及系统分析。结果引物Ps、OPLO4和引物组合P4，5可以得到布鲁氏菌的高分辨率的分型带谱，并建立相应的优化反应体系。结论在严格的优化反应条件下。建立简单、快捷、灵敏度高的RAPD方法，用于布鲁氏菌的DNA多态性研究，为布鲁氏菌分子流行病学的深入研究提供资料。     

Arbitrarily primed polymerase chain reaction fingerprinting for the genotypic identification of mutans streptococci from humans

Determining whether two strains of bacteria are unique, identical or clonally related depends upon comparisons of phenotypic and/or genotypic traits. Individual isolates can then be grouped according to differences or similarities among those traits. One method of genotyping strains of bacteria is commonly referred to as chromosomal DNA fingerprinting. Previously, we generated chromosomal DNA fingerprints of mutans streptococci to study the transmission of this organism within families. Here, we developed and evaluated an arbitrarily primed polymerase chain reaction (AP-PCR) method for the genotypic characterization of mutans streptococci. Results were compared to those derived from the more conventional chromosomal DNA fingerprinting method. First, we showed that randomly selected clinical isolates displayed a unique banding profile by both methods; the mean similarity indices between DNA fragment patterns were 0.69 for chromosomal DNA fingerprinting and 0.74 for AP-PCR. This indicated that AP-PCR demonstrated less diversity than chromosomal DNA fingerprinting. Subsequently, we tested the agreement between chromosomal DNA fingerprinting and AP-PCR in determining genotypic similarities among 21 mutans streptococci strains obtained from 10 mother-child pairs, and 5 mutans streptococci strains from 5 fathers. The Kappa value for agreement was 0.88. We conclude that AP-PCR, which generates patterns of 8 to 12 amplicons, is capable of distinguishing strains of mutans streptococci among non-related individuals. Moreover, AP-PCR can discern both homogeneity and heterogeneity of mutans streptococci genotypes among mother and child pairs. Overall, we found that AP-PCR gave results comparable to those of chromosomal DNA fingerprinting.     

Mutans streptococci strains prevalence before and after cavity preparation during Atraumatic Restorative Treatment

Critics argue that all carious dentine is not removed from the hand-prepared cavity during the Atraumatic Restorative Treatment (ART) procedure, and that the caries process is soon resumed. The aim of this study was to determine the effectiveness of ART in removing carious tissue, by investigating the numbers of mutans streptococci and lactobacilli, with emphasis on the prevalence of Streptococcus mutans and Streptococcus sobrinus strains before, and after ART treatment of dental caries. Two microbiology samples were collected. The first sample was removed from the centre of the carious lesion at the enamel-dentine junction, and the second was collected from the centre of the hard cavity wall above the pulp, after the soft infected dentine had been manually removed. A total of 71 mutans streptococci isolates from 31 children and 40 carious teeth were subcultured, biochemically characterised and genotyped by the arbitrarily primed polymerase chain reaction (AP-PCR). Results showed a significant decrease in TVC (P<0.0001), mutans streptococci (P < 0.0001) and lactobacilli (P = 0.0002) after cavity preparation. AP-PCR identified S. mutans strains that were undetectable during biotyping, and divided clinical isolates into two main clusters. In all, 63% (45/71) of isolates from the carious lesions comprised S. mutans strains. After cavity preparation, this was reduced to 35% (25/71), of which 30% (21/71) were S. mutans and the remaining 6% (4/71) S. sobrinus strains. The number of mutans streptococci strains was below detectable levels in 19 of the prepared cavities. The significant decrease in bacteria after manual cavity preparation demonstrates the reliability of a standardized ART technique, yet the presence of S. mutans strains shows that the effectiveness of the ART procedure can vary during treatment and between dental practitioners.