Use Of Restriction Endonuclease Analysis Research Articles

Antibacterial agents resistance and typing of β-lactamases in moraxella catarrhalis

目的 对卡他莫拉菌BROβ内酰胺酶进行分型分析,并对其与抗菌药物耐药的关系进行探讨.方法采用BSAC(british society for antimicrobial chemotherapy, BSAC)琼脂稀释法对本院呼吸道感染患儿鼻咽部分离的80株卡他莫拉菌株进行8种药敏试验,用Nitrocefin纸片检测β内酰胺酶,采用限制性内切酶分析方法进行bro 基因分型,同时将bro-1基因阳性和bro-2基因阳性菌株对β内酰胺类抗菌药物的耐药性进行比较.结果 (1) 80株卡他莫拉菌β内酰胺酶阳性率为95.0%.对氨苄西林MIC90为32 μg/ml,对头孢呋辛、头孢克洛和头孢噻肟的MIC90分别为4、8、1 μg/ml,对四环素的MIC90为16 μg/ml;环丙沙星为最敏感的抗菌药.(2)80株卡他莫拉菌bro基因分型结果:6株(7.5%)bro阴性,55株(68.8%)bro-1阳性,19株(23.8%)bro-2阳性.其中有2株bro基因阴性经Nitrocefin纸片法检测,β内酰胺酶结果为阳性,其他菌株基因检测与产酶结果一致.(3)bro-1阳性菌株对氨苄西林、头孢克洛的MIC90值与bro-2阳性菌株相比,有不同程度的升高.结论我国儿童携带的卡他莫拉菌产酶率高,对氨苄西林高度耐药.目前应加强该菌对头孢类和四环素的耐药监测.限制性内切酶分析方法在检测bro基因、筛选卡他莫拉菌对β内酰胺类抗生素的耐药及分子流行病学调查方面有一定的应用价值。

Typing of Candida krusei clinical isolates by restriction endonuclease analysis and hybridization with CkF1,2 DNA probe.

The use of restriction endonuclease analysis and Southern hybridization with our new CkF1,2 DNA probe, cold labeled with peroxidase, for the typing of Candida krusei isolates has been investigated. Fifty-five clinical samples isolated from forty-five patients hospitalized in eight centers, one environmental strain, and two reference strains were evaluated. Patterns were analyzed by a computer-assisted method and compared by numerical analysis. Clearer and less ambiguous patterns were obtained by restriction with endonuclease HinfI. It generated 9 to 14 (average, 11) well-separated fragments in the range of 6.5 to 2.0 kb. Both their numbers and sizes varied greatly among the strains studied. The CkF1,2 probe hybridized with one to seven fragments of HinfI patterns. A total of 48 distinct types were distinguished among the 58 strains studied. HinfI and CkF1,2 patterns showed similarities of less than 83 and 75% for unrelated strains and more than 91 and 100% for related strains, respectively. The methods showed 100% typeability, 98% reproducibility, and a discriminatory power of 1. C. krusei isolates from each patient were distinct, whether from one hospital or from different hospitals. Multiple isolates from the same patient were identical, both over time and at different anatomic sites. An endogenous origin is suggested for the colonizing and infecting isolates among the 45 patients. The CkF1,2 probe enhanced discrimination of the strains and provided a definitive comparison for strain identity. Genetic linkages between isolates were assessed at the subspecies level, and 12 clusters were delineated. A typing scheme is proposed for epidemiological studies of C. krusei.

Investigation of an epidemic of invasive aspergillosis

When seven immunocompromised patients developed invasive aspergillosis during construction at a hospital, new methods were performed to compare fungal isolates and a case-control study was conducted to determine risks for infection. Typing of Aspergillus flavus with the use of restriction endonuclease analysis and restriction fragment length polymorphism using random amplified polymorphic DNA reactions to generate DNA probes revealed different patterns between isolates from two patients and a similar pattern among those from one patient, a health care worker, and an environmental source. Case patients were more likely than controls to have longer periods of hospitalization (median, 83 vs. 24 days; P < 0.01), neutropenia (median, 33 vs. 6 days; P < 0.05), and exposure to broad spectrum antimicrobials (median, 56 vs. 15 days; P = 0.08). No patients restricted to protected areas developed aspergillosis. Risk of exposure of immunocompromised patients to opportunistic organisms stirred up by construction activity may be decreased by admitting these patients to protected areas away from construction activity and by restricting traffic from construction sites to these areas. Although typing of A. flavus isolates did not reveal a single type or source of organism responsible for infection, this method may facilitate epidemiologic investigation of possible nosocomial sources and transmission in similar settings.

Molecular analysis of multiple isolates of the major serotypes of group B streptococci.

Serotyping of clinical isolates is a widely used technique for epidemiologic study of group B streptococcal infections. However, serotyping cannot definitively determine epidemiologically related or unrelated isolates. We investigated the use of restriction endonuclease analysis (REA) with both conventional agarose gel electrophoresis (AGE) and pulsed-field gel electrophoresis (PFGE) in 50 isolates of the major serotypes of group B streptococci. Single digestion with HindIII and HaeIII and double digestion with HindIII and then EcoRI were used for conventional AGE, and digestion with SmaI was used for PFGE. The molecular profile of one strain was compared with those of the strains within the same serotype as well as with the profiles from strains of different serotypes. Among 10 type Ia, Ia/alpha, Ia/alpha+beta, and Ia/R1 isolates and depending on the restriction enzyme used, we found between five and six REA patterns by conventional AGE and seven by PFGE; among 4 type Ib/alpha+beta isolates we found 2 to 4 REA patterns by conventional AGE and 4 by PFGE; among 21 type II, II/alpha, II/beta, II/alpha+beta, and II/R4 isolates, we found 11 REA patterns by both AGE and PFGE; and among 14 type III, III/R1, and III/R4 isolates, we found from 7 to 12 different REA patterns by AGE and 10 by PFGE. In total, among 13 serotypes and one nontypeable strain, we found 29 to 31 REA patterns by conventional AGE and 33 by PFGE. A particular REA pattern within a serotype was different from the patterns found in the other serotypes, suggesting that REA analysis by using conventional AGE or PFGE is a sensitive method for analyzing genetic relatedness and diversity in group B streptococci and has potential value in molecular epidemiologic studies.

Subtyping of a frequent phagovar of Listeria monocytogenes in Sweden by use of restriction endonuclease analysis.

In Sweden, many Listeria monocytogenes strains belonging to serovar 4b and isolated during the last five years from different sources share the same phagovar--2389:2425:3274:2671:47:108:340. The object of the present study was to investigate if 31 L. monocytogenes serovar 4b strains belonging to this particular phagovar could be differentiated by use of a simple restriction endonuclease analysis (REA). Among the enzymes tested, Xho I was found to be the most useful, since this enzyme could divide the 31 strains into five groups. The profiles of all human clinical isolates were indistinguishable from each other, which indicates that these strains may represent a single clone. The food isolates and the strains of human origin did not share the same profile. This further characterization may be of epidemiological importance as this phagovar of L. monocytogenes has been associated with at least two outbreaks of human listeriosis in Europe.

An investigation into the use of restriction endonuclease analysis for the study of transmission of Actinomyces

DNA fingerprints of 28 reference strains of Actinomyces, comprising representatives of different species and serotypes, and 19 isolates recovered from 16 periodontal patients was performed. The aim was to determine the potential of the method for detecting strain differences in terms of discriminatory power and to evaluate its usefulness in the typing of Actinomyces strains for eco-epidemiological studies. Among the 17 restriction endonucleases tested, Bst EII, Pvu II and Sma I proved to be the most suitable for the genus Actinomyces restriction digest analysis. Visual comparisons of Bst EII, Pvu II and Sma I digest patterns of chromosomal DNA revealed clear differences within species but also within serotypes of Actinomyces that are otherwise identical. The method offers the qualities for use as an epidemiological tool for identifying sources and tracing routes of transmission of Actinomyces: stability, reproducibility, ease of preparation and interpretation and enough sensitivity for detection of differences between morphologically and serologically similar strains of Actinomyces.

Molecular epidemiology of group B streptococcal infections: use of restriction endonuclease analysis of chromosomal DNA and DNA restriction fragment length polymorphisms of ribosomal RNA genes (ribotyping).

Epidemiologic investigation of group B streptococcal (GBS) infections has been limited by the lack of a discriminatory typing system. Therefore, the use of restriction endonuclease analysis of chromosomal DNA (REAC) and DNA restriction fragment length polymorphisms of rRNA genes (ribotyping) to subtype molecularly GBS isolates associated with human invasive disease was investigated. Chromosomal DNA of selected GBS isolates was initially digested with 24 different restriction enzymes. HhaI gave the best discrimination of hybridization banding patterns (ribotypes) and was used with all study isolates. Ribotyping and REAC differentiated among isolates of the same and different serotypes. Nine ribotype patterns were noted among the 76 isolates studied, including 4 among serotype Ia/c and 4 additional ribotypes among serotype III isolates. Epidemiologically related isolates (e.g., mother-infant or twin-twin pairs) had identical REAC and ribotype patterns. Epidemiologically unrelated isolates with the same ribotype usually had different REAC patterns, suggesting that REAC may be a more sensitive technique for strain differentiation. REAC and ribotyping were reproducible and proved to be successful molecular epidemiologic methods for subtyping GBS.

Abortifacient property of bovine herpesvirus type 1 isolates that represent three subtypes determined by restriction endonuclease analysis of viral DNA

SUMMARY Bovine herpesvirus type 1 (bhv-1) isolates are classified into 3 subtypes by use of restriction endonuclease analysis. Isolates from aborted fetuses have been either subtype 1 or 2a, whereas subtype 2b viruses have not been associated with abortion. We assessed the abortifacient property of isolates representing each of the 3 bhv-1 subtypes by iv inoculation of heifers with the virus 25 to 27 weeks after breeding. Three heifers were given Cooper (subtype 1) isolate, 3 heifers were given FI (subtype 2a) isolate, and 5 heifers were given K22 (subtype 2b) isolate. All heifers developed fever and viremia 2 to 5 days after inoculation. Heifers given Cooper or FI isolate aborted between 17 and 85 days after inoculation. The 5 heifers given K22 isolate delivered full-term calves. Placenta was obtained from 4 of the 5 heifers, and K22 virus was isolated from each placenta. Four calves had bhv-1 neutralizing antibody in precolostral serum, with titer ranging from 1:4 to 1:512.

An investigation into the use of restriction endonuclease analysis for the study of transmission of mutans streptococci.

Restriction endonuclease analysis (REA) was performed on the total cellular DNA from each of 396 strains of mutans streptococci (1) to determine its potential usefulness for the study of transmission of the organism and (2) to document the proportions and variety of strains harbored by members of a small group of families. The DNA was digested with restriction enzyme EcoRI and/or HindIII, electrophoresed on agarose gels, and the resulting patterns compared. The strains examined included fresh isolates from 58 subjects, including 19 strains from each member of five families. The sensitivity and reproducibility of REA patterns from the mutans streptococci seemed ideal for studies of their epidemiology and transmission. The pattern of each isolate from humans was unique, except for isolates from the same individual or from the same family. REA types from subjects from different families were always heterogeneous. A high frequency of multiple REA types (up to 5) was observed in many subjects. While evidence for intra-familial transmission was obtained, including transmission between spouses, there was also strong evidence of frequent sources of infection outside of the family. Mutations of strains to streptomycin resistance or to lactate dehydrogenase deficiency caused no detectable change in the REA patterns. The lack of plasmids in any of the 57 fresh isolates that were examined for them suggested that they may have contributed little to the heterogeneity of the patterns seen.

Plasmid Fingerprinting: A Tool for Bacterial Strain Identification and Surveillance of Nosocomial and Community-Acquired Infections

Plasmid fingerprinting provides a rapid and dependable means of identifying bacterial isolates of the same strain. The stability, wide distribution, and diverse nature and size of extrachromosomal elements make it suitable for virtually all bacterial genera. There are many different procedures available for plasmid screening, and the one chosen depends primarily on the types of organisms to be analyzed. Some procedures are better suited to gram-positive organisms; others are better for visualizing the very large plasmids often seen in Pseudomonas and Rhizobium species. The key to the plasmid fingerprinting technique is agarose gel electrophoresis. In this step of the technique, it is important to differentiate open circular from closed circular forms of plasmids and to recognize the "smile effect." Plasmid fingerprinting can be utilized for epidemiologic studies of both nosocomial and community-acquired infections. The use of restriction endonuclease analysis can greatly enhance the ability of the investigator to differentiate strains that harbor only a single plasmid. Plasmid fingerprinting often provides the only differential characteristic for strains involved in epidemics.