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PubMed1991

Identification of epidemic strains of Streptococcus suis by genomic fingerprinting.

A natural outbreak of Streptococcus suis meningitis in two closed swine herds was studied. DNA fingerprinting, serotyping, and biochemical profiles were assessed. Multiple serotypes were recovered from these herds. In farm A, 50 S. suis strains were isolated from 330 swabs collected. Eighteen strains belonged to serotype 2, and 32 strains belonged to serotypes 3, 5, 6, 8, 9, and 11. In farm B, 16 S. suis strains were recovered from a total of 70 samples. Eight strains belonged to serotype 7 and eight belonged to serotypes 2, 3, 5, and 8. In each epidemiological situation, a single strain characterized by a distinctive restriction fragment pattern predominated among affected penmates. The epidemic serotype 2 strain was detected in farm A in weaned pigs between the ages of 5 and 7 weeks. In contrast, the pathogenic strain in farm B belonged to serotype 7 and was isolated from pigs up to 3 weeks of age. The results from both farms strongly suggest a lateral spread of these organisms. No vertical transmission could be shown in either herd. It was concluded that genomic fingerprinting is an appropriate method to distinguish outbreak isolates of S. suis from nonoutbreak strains, within the same serotype or from epidemiologically unrelated clusters of strains.

Animals↗
PubMed1991

DNA fingerprinting and electrophoretic karyotype of environmental and clinical isolates of Candida parapsilosis.

The endonuclease restriction pattern (DNA fingerprinting) and the electrophoretic karyotype of 16 Candida parapsilosis isolates from environmental and clinical sources were investigated. DNA from both whole cells and separated mitochondria was digested with enzymes, including EcoRI, BamHI, KpnI, BglII, HpaII, PvuII, and HindIII. Regardless of their source and pathogenic properties, all isolates showed a uniform, reproducible, and overlapping whole-cell DNA fingerprinting with each endonuclease digest. Mitochondrial DNA fragments were, in all cases, major contributors to the total cellular DNA restriction pattern. In contrast, the electrophoretic karyotype generated by rotating field gel electrophoresis (RFGE) or contour clamped homogeneous field electrophoresis (CHEF) showed a remarkable polymorphism among the isolates. This polymorphism concerned the smaller molecular size section of the karyotype (range, 1.8 to 0.7 Mb), where at least two to five chromosomal bands could be consistently detected by both RFGE and CHEF. Larger (greater than or equal to 3.0 to 1.9 Mb) chromosome-sized DNA bands (four in CHEF and three in RFGE) were quite distinct and common to all isolates. Thus, seven karyotype classes could be defined, on the basis of both the number and size of putative chromosomes. The three categories of isolates (soil, vaginal, and hematological) were not randomly distributed among the seven classes. In particular, the four hematological isolates had a karyotype pattern which was clearly distinct from that shown by the three environmental isolates, and of the nine vaginal isolates only one shared a class with isolates from another source (soil). Although tentative, the classification was totally consistent with the independent and reproducible results obtained by the two pulse-field electrophoretic techniques employed. It is suggested that the electrophoretic analysis of the karyotype might be particularly useful for epidemiological and pathogenicity studies on biotypes of C. parapsilosis.

Candida↗
PubMed1991

DNA fingerprinting of isolates of Staphylococcus aureus from newborns and their contacts.

During a study on the epidemiology of Staphylococcus aureus colonization in newborns, mothers, and hospital staff, S. aureus was isolated from 536 of 1,945 specimens. Ninety-three isolates of S. aureus from the three groups of individuals were included in a study to evaluate the potential of DNA fingerprinting for strain differentiation. The 93 isolates were also phage typed and their plasmid profiles were analyzed. Cleavage of DNA with BamHI resulted in 13 different DNA restriction endonuclease band patterns (DNA REBPs), one of which consisted of eight isolates whose DNA was not cleaved with BamHI. The DNAs from these eight isolates were easily cleaved with HindIII. The different DNA REBPs were stable both during in vitro and in vivo growth and allowed strain differentiation within phage groups or types. We could not show any strong association between DNA REBP classes, phage types or groups, and plasmid profiles. Of the 93 isolates, 27 (29.0%) could not be phage typed and 12 (12.9%) lacked plasmids. We therefore conclude that DNA fingerprinting is a powerful tool, in addition to phage typing and plasmid profile analysis, for strain differentiation of S. aureus.

Adult↗
PubMed1992

DNA fingerprinting by pulsed-field gel electrophoresis is more effective than ribotyping in distinguishing among methicillin-resistant Staphylococcus aureus isolates.

Pulsed-field gel electrophoresis (PFGE) after SmaI restriction of DNA from 239 methicillin-resistant Staphylococcus aureus isolates (from 142 patients) produced 26 different fingerprints. The deduced chromosome sizes ranged from 2,200 to 3,100 kb (+/- 100 kb). A total of 81 isolates taken from 65 patients were then typed by PFGE and ribotyping with ClaI, EcoRI, and HindIII. Ribotypes were less discriminating than PFGE. Ribotyping did not discriminate isolates from a given PFGE fingerprint into different subsets. PFGE may be a more effective epidemiological tool than ribotyping for the typing of methicillin-resistant S. aureus strains.

Bacterial Typing Techniques↗
PubMed1992

Subtyping of Streptococcus uberis by DNA amplification fingerprinting.

Total DNA of Streptococcus uberis from cows with mastitis was analyzed by DNA amplification fingerprinting (DAF) and compared with restriction endonuclease fingerprinting (REF). DAF grouped 22 strains into 15 distinct patterns, while REF grouped them into 12 patterns. These results suggest that DAF is a useful technique for subtyping strains of S. uberis.

Base Sequence↗
PubMed1993

Epidemiological fingerprinting of Enterobacter cloacae by small-fragment restriction endonuclease analysis and pulsed-field gel electrophoresis of genomic restriction fragments.

A cluster of infections caused by Enterobacter cloacae was observed among preterm neonates in a neonatal intensive care unit (NICU) of a pediatric hospital in Osnabrück, Germany. The presence of similar antimicrobial susceptibility patterns among the bacterial isolates prompted an investigation to determine whether a limited spread of a single strain existed. All 12 E. cloacae isolates from the NICU and 50 nonrelated strains were fingerprinted by small-fragment restriction endonuclease analysis (SF-REA) of EcoRI DNA digests. Selected isolates were further characterized by pulsed-field gel electrophoresis (PFGE) of NotI- or XbaI-generated genomic restriction fragments. Epidemiologically unrelated strains were clearly discriminated by both methods. Results achieved by SF-REA and PFGE revealed that of the 12 isolates from the NICU, 11 belonged to the same genotypic cluster. Since all reagents and equipment for both techniques are commercially available, DNA fingerprinting by SF-REA or PFGE is proposed as a useful tool in the microbiology laboratory for investigating the epidemiological relatedness of E. cloacae strains of clinical and environmental origin.

Cross Infection↗
PubMed1993

Genomic fingerprinting of "Haemophilus somnus" isolates by using a random-amplified polymorphic DNA assay.

The random-amplified polymorphic DNA (RAPD) assay was used to generate DNA fingerprints for 16 isolates of "Haemophilus somnus," and one isolate each of "Haemophilus agni," "Histophilus ovis," "Actinobacillus seminis," Pasteurella haemolytica, and Escherichia coli. The RAPD assay differentiated among "H. somnus" isolates, which shared similarity coefficients of 0.46 to 1.00 on the basis of pairwise comparisons of RAPD markers produced with nine random decamer primers. Three virulent encephalitic "H. somnus" isolates exhibited identical banding patterns, suggesting a common clonal ancestry. The RAPD assay clearly distinguished between the "H. somnus"-"H. agni"-"H. ovis" group and the other bacterial species tested. The results of the present study suggest that DNA fingerprinting of "H. somnus" isolates by the RAPD assay could be valuable in revealing subspecific divisions within this largely unexplored species.

Base Sequence↗
PubMed1993

Mixed-linker polymerase chain reaction: a new method for rapid fingerprinting of isolates of the Mycobacterium tuberculosis complex.

Rapid recognition of multidrug-resistant strains of Mycobacterium tuberculosis is a desirable goal for treatment of patients and protection of health care workers. DNA fingerprints produced with the insertion sequence IS6110 generate restriction fragment length polymorphism (RFLP) patterns that reliably identify M. tuberculosis complex strains. This report describes a rapid technique for RFLP typing using the polymerase chain reaction. The method uses one primer specific for IS6110 and a second primer complementary to a linker ligated to the restricted genomic DNA. In one strand the linker contains uracil in place of thymidine, and specific amplification is obtained by elimination of this strand with uracil N-glycosylase. Mixed-linker fingerprinting clearly differentiated multidrug-resistant isolates from 12 outbreaks and unambiguously assigned them to 26 RFLP groups.

Base Sequence↗
PubMed1993

Rapid fingerprinting of Helicobacter pylori by polymerase chain reaction and restriction fragment length polymorphism analysis.

A simple and reliable technique was developed for differentiating Helicobacter pylori strains by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified DNAs. Oligonucleotide primer pairs developed to the urease, 48-kDa stress protein (htrA), and 26-kDa antigen-encoding genes were used to amplify fragments of the appropriate size from crude boiled cell preparations. The PCR-amplified products were digested with Sau3A, HaeIII, MspI, AluI, MluI, HinfI, and XbaI restriction endonucleases. Restriction fragment length polymorphisms were particularly evident within the urease and htrA genes and were easily detected by Sau3A, HaeIII, MspI, and AluI restriction endonuclease analysis. Double digestion of these separately amplified products or restriction analysis of multiple PCR-amplified fragments was found to discriminate 17 of 17 (100%) H. pylori strains which had unique genomic DNA fingerprints. Results of an investigation of multiple isolate sets obtained from patients before and after therapy was consistent with the hypothesis that treatment failures were due to the persistence of the same strain but did not discount the possibility that the patients were reinfected with a strain shared by family members or close contacts. The results indicate that the PCR-restriction endonuclease analysis method can be applied directly to biopsy samples, has the potential to fingerprint H. pylori isolates rapidly, and may permit detailed epidemiological investigations on the transmission of this important pathogen.

Base Sequence↗
PubMed1993

Development of DNA probes for fingerprinting Aspergillus fumigatus.

Several different DNA fragments containing nonribosomal repetitive sequences have been isolated from the genome of Aspergillus fumigatus and tested as potential DNA fingerprinting probes. Eight of these clones generate 19 or more bands when hybridized to EcoRI-digested DNA of a reference strain in Southern blots, and they fall into four families. Individual clones from two families were tested and were found to generate complex Southern blot hybridization patterns which are stable within a single strain over many generations, which vary among unrelated strains, and which are amenable to computer-assisted analyses involving large numbers of strains in epidemiological studies. Clones from three of the families clustered a majority of test strains in a similar fashion in individual dendrograms based on similarity coefficients computed from band positions in Southern blot hybridization patterns. These clones therefore fulfill the major requisites for effective DNA fingerprinting probes.

Aspergillosis↗
PubMed1993

Epidemiological study of an Acinetobacter baumannii outbreak by using polymerase chain reaction fingerprinting.

A polymerase chain reaction (PCR) technique was applied to the fingerprinting of different strains of Acinetobacter baumannii from a cluster of patients infected or colonized with the incriminated pathogen. The DNA was extracted by boiling and was subjected to PCR amplification by using the core sequence of the M13 phase as a single primer. The amplified products were separated by agarose gel electrophoresis and were detected by staining with ethidium bromide. In 1990, 49 multiresistant A. baumannii strains were isolated from 13 patients from the same intensive care unit of the Charité Hospital; 45 of these outbreak isolates obtained from 12 patients showed the same PCR patterns, indicating an epidemiological relatedness of these strains. Four strains isolated from the same patient belonged to another genetic group, as revealed by a distinct amplification pattern. Another single subtype of A. baumannii was identified as the causative agent in patients during a second outbreak at a different intensive care unit in the same hospital. Seventeen isolates recovered from 10 immunocompromised patients had the same amplification patterns, which were distinct from all other PCR profiles. Five strains were obtained from two other hospitals; three isolates from the hospital of Magdeburg, Germany, had identical PCR patterns which, however, could be clearly distinguished from the patterns of all other strains. The remaining two isolates displayed individual patterns of amplified fragments. PCR fingerprinting may provide a useful and particularly rapid identification technique for epidemiological investigations of nosocomial infections.

Acinetobacter↗
PubMed1993

Large-scale DNA fingerprinting of Mycobacterium tuberculosis strains as a tool for epidemiological studies of tuberculosis.

We conducted a large-scale DNA fingerprinting analysis of Mycobacterium tuberculosis strains in a country in which tuberculosis is endemic (Tunisia) in order to evaluate the importance of microepidemics in the maintenance of the disease within the population. The genetic polymorphisms of 201 strains of M. tuberculosis isolated from 196 unrelated patients living in four districts of northern Tunisia during a 3-year period were studied by restriction fragment length polymorphism (RFLP) analysis by using the insertion sequence IS6110 as a probe. Seventy-three strains isolated from 68 patients living in the districts of Tunis, Nabeul, and Jendouba generated 67 different RFLPs, indicating a high degree of polymorphism of the M. tuberculosis strains within these areas. In contrast, the 128 strains isolated from individuals in the district of Menzel Bourguiba appeared much less heterogeneous since they often generated identical or very similar fingerprints. Seventeen of 29 cases (58%) of active tuberculosis in the city of Menzel Bourguiba could be traced to as few as four M. tuberculosis strains. These results indicate the persistence of underestimated microepidemics in this region. The RFLP typing of a large number of randomly collected strains provides a general picture of the strains involved in tuberculosis. The systematic study of limited areas where tuberculosis is endemic can provide evidence for the existence of persisting epidemics. This stresses the different problems which remain to be solved in order to improve the control of tuberculosis.

DNA Fingerprinting↗
PubMed1993

Genomic fingerprinting of Neisseria meningitidis associated with group C meningococcal disease in Canada.

A single electrophoretic type (ET15) of Neisseria meningitidis has been associated with an increased incidence of group C meningococcal disease in Canada. Genomic fingerprinting through pulsed-field gel electrophoresis of chromosomal DNA was used to characterize the clonal relationship among meningococcal isolates of different electrophoretic types and among isolates within ET15. The genomic fingerprints of the ET15 isolates, while similar as a group, were sufficiently distinct to confirm linkage for four pairs of strains from focal outbreaks and differed markedly from those of the other common electrophoretic types, ET5, ET9, and ET21.

Canada↗
PubMed1994

Plasmid DNA fingerprinting of Acinetobacter species other than Acinetobacter baumannii.

During the last years Acinetobacter species have emerged as clinically significant pathogens. Most infections are nosocomially acquired and mainly due to Acinetobacter baumannii. Little is known about the epidemiology and clinical significance of unnamed Acinetobacter species 3 (the second most often encountered member of the genus Acinetobacter) and other Acinetobacter species such as A. johnsonii, A. junii, and A. lwoffii. Seventy-five clinical isolates of Acinetobacter species other than A. baumannii (Acinetobacter species 3, n = 37; A. johnsonii, n = 20; A. junii, n = 8; A. lwoffii, n = 10) recovered from 66 patients over a period of 12 months were analyzed by plasmid DNA fingerprinting. Plasmids were found in 84.4% of Acinetobacter species 3 isolates and in all A. johnsonii, A. junii, and A. lwoffii isolates. Strains harbored up to 15 plasmids each. Almost every isolate gave a unique plasmid pattern. With one exception, identical plasmid profiles were detected only in corresponding isolates recovered from blood cultures and intravascular catheters from a given patient. Plasmid DNA fingerprinting proved to be useful for typing Acinetobacter species other than A. baumannii. There was no evidence of patient-to-patient transmission or hospital outbreaks due to these species. This finding is in contrast to the results obtained in studies of the hospital epidemiology of A. baumannii.

Acinetobacter↗
PubMed1994

Typing of Mycoplasma pneumoniae by PCR-mediated DNA fingerprinting.

PCR fingerprinting was used to characterize clinical isolates of Mycoplasma pneumoniae. Among 24 strains tested, two types were distinguished. Nineteen strains belonged to type 1, whereas only 5 strains belonged to type 2. The majority of strains isolated since 1991 in Belgium belong to type 1. No variations in fingerprinting patterns were observed within each group, confirming the highly conserved nature of the M. pneumoniae genome.

Bacterial Typing Techniques↗
PubMed1995

Long-term colonization with single and multiple strains of Helicobacter pylori assessed by DNA fingerprinting.

The gastric pathogen Helicobacter pylori establishes long-term chronic infections that can lead to gastritis, peptic ulcers, and cancer. The species is so diverse that distinctly different strains are generally recovered from each patient. To better understand the dynamics of long-term carriage, we characterized H. pylori isolates from initial and follow-up biopsy specimens from a patient population at high risk of H. pylori infection and gastric cancer. Eighty-five isolates were obtained from 23 patients and were analyzed by genomic restriction enzyme analysis, arbitrarily primed PCR fingerprinting, (random amplified polymorphic DNA analysis), and/or restriction of specific PCR-amplified genes (restriction fragment length polymorphism analysis). A single strain was found in sequential biopsy specimens from 12 of 15 patients (80%) receiving sucralfate. In the remaining three patients treated with sucralfate, two strains were identified in two patients and three strains were identified in the third patient. In contrast, a single strain was found in sequential biopsy specimens from only three of eight patients (37%) receiving bismuth, metronidazole, and nitrofurantoin. Two strains were identified in five other patients receiving bismuth-antibiotic (63%). Immunoglobulin G antibodies to H. pylori were present in the sera of all patients. Thus, H. pylori colonization can persist for long periods (up to at least 4 years), despite high titers of immunoglobulin G antibodies in serum. Resistance to metronidazole was noted in some strains before and/or after treatment, but all strains remained susceptible to amoxicillin, tetracycline, and nitrofurantoin. We conclude that H. pylori genotypes, as measured by several sensitive DNA fingerprinting methods, can remain stable for years in vivo, despite the acquisition or loss of drug resistance, circulating antibody, or exposure to antibiotics or sucralfate.

Anti-Bacterial Agents↗
PubMed1995

Evidence for nosocomial transmission of Candida albicans obtained by Ca3 fingerprinting.

The moderately repetitive sequence Ca3 was used to fingerprint Candida albicans isolates from 32 patients hospitalized for more than 3 days, 17 recent admissions or outpatients, and 8 recently readmitted patients and 10 commensal isolates from the community in Wellington, New Zealand, plus isolates from 21 hospitalized patients, 26 outpatients or recent admissions, 4 recently readmitted patients, and 10 healthy individuals in the community in Auckland, New Zealand. In Wellington, isolates from patients hospitalized in Wellington Hospital for more than 3 days were genetically significantly less diverse than were isolates from outpatients or recent admissions or isolates from healthy individuals in the community. In addition, two clusters of genetically similar strains were isolated from hospitalized patients significantly more often than from other individuals. These observations provide evidence (albeit indirectly) for nosocomial transmission of hospital-specific C. albicans strains. In contrast, no indication of hospital-specific transmission of C. albicans was found in Auckland Hospital. Since these results were obtained under conditions in which no candidiasis outbreak occurred in either hospital, they also suggest that Ca3 fingerprinting may be a useful tool in preventive nosocomial infection control programs, allowing assessment of the extent of C. albicans transmission occurring in a hospital.

Adolescent↗
PubMed1995

Colonizing populations of Candida albicans are clonal in origin but undergo microevolution through C1 fragment reorganization as demonstrated by DNA fingerprinting and C1 sequencing.

The genetic homogeneity of nine commensal and infecting populations of Candida albicans has been assessed by fingerprinting multiple isolates from each population by Southern blot hybridization first with the Ca3 probe and then with the 0.98-kb C1 fragment of the Ca3 probe. The isolates from each population were highly related, demonstrating the clonal origin of each population, but each population contained minor variants, demonstrating microevolution. Variation in each case was limited to bands of the Ca3 fingerprint pattern which hybridized with the 0.98-kb C1 fragment. The C1 fragment was therefore sequenced and demonstrated to contain an RPS repetitive element. The C1 fragment also contained part or all of a true end of the RPS element. These results, therefore, demonstrate that most colonizing C. albicans populations in nonimmuno-suppressed patients are clonal, that microevolution can be detected in every colonizing population by C1 hybridization, and that C1 contains the repeat RPS element.

Base Sequence↗