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PubMed1989

Plasmid DNA analysis by agarose gel electrophoresis on an epidemic-associated strain of trimethoprim resistant Shiga's bacillus from Gimira Wereda (Keffa administrative region, southwest Ethiopia).

A combination of genetic mating experiments and plasmid DNA analysis by gel electrophoresis was carried out on an epidemic-associated Shiga's bacillus with resistance type ACSSuTTp, isolated in 1983 from Gimira Wereda, Keffa Administrative Region, South West Ethiopia. The donor strain contained 9 plasmid species. Their sizes in megadaltons (Mdal) were: 120, 40, 35, 12.5, 6.0, 5.1, 4.3, 2.5 and 1.7. Analysis of Escherichia coli K12 transconjugants revealed that the 40, 35, and 4.3 Mdal plasmids coded for tetracyline-trimethoprim, ampicillin -chloramphenicol and streptomycin-sulphadiazine resistance respectively.

DNA, Bacterial↗

PubMedDate not supplied

Application of genomic DNA subtyping by pulsed field gel electrophoresis and restriction enzyme analysis of plasmid DNA to characterize methicillin-resistant Staphylococcus aureus from two nosocomial outbreaks.

Pulsed-field gel electrophoresis (PFGE) and restriction enzyme analysis of plasmid DNA (REAP) were applied to study the epidemiologic relationship among methicillin-resistant Staphylococcus aureus (MRSA) isolates from outbreaks in two hospitals in São Paulo, Brazil: 82 MRSA isolates, 73 from a university hospital and nine from a general adult intensive care unit of a private hospital, were collected from 62 patients: 95% of the MRSAs were also resistant to gentamicin and ciprofloxacin. REAP subtyping of both collections identified six different subtypes: 55 (72.6%) MRSAs from the university hospital and nine isolates from the private hospital shared the same epidemic REAP subtype. Discrimination by restriction of genomic DNA with Sma I followed by PFGE enabled the identification of 14 DNA subtypes. Based on the combined REAP-genomic DNA subtype, the predominant subtype in the university hospital was A/A (44 isolates) whereas the epidemic subtype in the private hospital was A/M (seven isolates). The application of two typing methods showed better discrimination among MRSAs than did either method alone.

Bacterial Typing Techniques↗

PubMed1998

The epidemiology of Salmonella typhimurium in cattle: plasmid profile analysis of definitive phage type (DT) 204c.

During the period 1979-1991, Salmonella Typhimurium DT 204c was the cause of a major epidemic of salmonellosis in calves in the UK. Plasmid profile analysis of DT 204c isolates from England and Wales commenced in 1986 and isolates from all subsequent incidents were examined by this technique. Forty-three different plasmid profile types (PPTs) were detected, of which the commonest, designated type E, constituted 44.6-80.2% of the annual incidents during the study period. Some PPTs, e.g., F and P, were detected throughout most years of the study, whereas PPTs O and 6 persisted for short periods. Until 1984, most isolates were resistant to neomycin, but the subsequent predominant PP type E was sensitive to this antibacterial agent. It was concluded that during the epidemic there was an evolution of new genotypes, of which only some persisted; again, antibacterial resistance genes may be acquired or lost. The study demonstrated the value of PP typing for epidemiological studies.

Animals↗

PubMed1996

The use of plasmid profile analysis and ribotyping for typing Acinetobacter baumannii isolates.

Plasmid profiles were used to analyse 39 Acinetobacter baumannii isolates from 36 patients at three hospitals. The isolates were prevously classified by biotyping and rDNA fingerprinting. Ribotyping was useful to establish the lineage of isolates and to confirm genospecies identification. Thirty-seven isolates (94.9%) contained plasmids. The variable number of plasmids with different molecular weights in each isolate enabled the identification of 13 profiles without the need for endonuclease digestion. Fifteen A. baumannii biotype 2 isolates of similar ribotype and antibiotype contained identical plasmids over a two-month outbreak at one hospital. Plasmid typing discriminated these isolates from sporadic A. baumannii isolates of close ribotype obtained from different hospitals. A few isolates of different lineage, however, showed similar plasmid profile. Our results suggest that plasmid typing is a practical method to assist infection control of nosocomial A baumannii. A combination of plasmid typing and ribotyping is suggested to confirm genospecies classification and to identify strains against reference band profiles.

Acinetobacter↗

PubMed1990

Evaluation of restriction endonuclease fingerprinting of chromosomal DNA and plasmid profile analysis for characterization of multiresistant coagulase-negative staphylococci in bacteremic neonates.

A procedure was developed for restriction endonuclease fingerprinting (REF) of the chromosomal DNA of coagulase-negative staphylococci. A total of 48 isolates comprising 29 Staphylococcus epidermidis and 19 Staphylococcus haemolyticus isolates from blood and mucocutaneous sites of 15 premature neonates were characterized by REF, plasmid profile (PP) analysis, antimicrobial susceptibility testing, biotyping, and slime production. On the basis of REF analysis of chromosomal DNA, the 48 coagulase-negative staphylococcal isolates were subdivided into 10 subgroups, whereas PP analysis subdivided the strains into 20 distinct subgroups. REF analysis of total DNA (i.e., chromosome plus plasmid) resulted in the same 20 subgroups as were subdivided by PP analysis. The high discriminatory power of PP analysis was associated with the variability of plasmid content in coagulase-negative staphylococcal strains isolated during the outbreak. REF patterns were found to be stable both in vitro and in vivo. Isolates carried from 2 to 10 plasmids that ranged in molecular size from 0.9 to 39.5 megadaltons. Plasmids were disseminated among the coagulase-negative staphylococci, regardless of the genetic relatedness of their chromosomal DNAs. Hence, a lack of correlation existed between the grouping of isolates by REF analysis of chromosomal DNA and the grouping by PP analysis. There were one and two distinct chromosomal patterns among 4 of 4 blood cultures and 15 of 15 mucocutaneous cultures of S. haemolyticus, respectively. In contrast, a higher proportion of distinct chromosomal patterns was found for S. epidermidis in blood cultures (7 of 11 cultures) compared with those identified for isolates in mucocutaneous cultures (6 of 18 cultures). In summary, REF analysis of chromosomal DNA, rather than total DNA, is a useful marker for epidemiological investigations of coagulase-negative staphylococci. PP analysis can also be used to provide additional epidemiological information regarding the most recent genetic events.

Coagulase↗

PubMed2001

Analysis of Salmonella enteritidis strains isolated from food-poisoning cases in Taiwan by pulsed field gel electrophoresis, plasmid profile and phage typing.

AIMS: To establish the molecular typing data for Salmonella enteritidis due to its increasing role in Salmonella infections in Taiwan. METHODS AND RESULTS: Sixty-three Salm. enteritidis strains isolated from related and unrelated patients suffering from food-borne poisoning during 1991-97 were collected and subjected to pulsed field gel electrophoresis (PFGE), plasmid analysis and phage typing. For PFGE, XbaI, SpeI and NotI restriction enzymes were used for chromosomal DNA digestion. The results showed that, for these 63 Salmonella strains, 10 PFGE pattern combinations were found. Of these, pattern X3 S3 N3 was the major subtype, since 46 strains isolated from different locations at different times during 1991-97 showed this PFGE pattern. Plasmid analysis showed only three plasmid profiles and phage typing showed that most of the Salmonella strains were of the phage type PT4. CONCLUSION: Most of the Salm. enteritidis strains circulating in Taiwan are of very similar genetic types or are highly related and that strains of PFGE pattern X3 S3 N3 are the prevalent and recirculating strains of Salm. enteritidis which caused food-poisoning cases in Taiwan in 1991-97. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information that in Salmonella infection, certain subtypes of Salm. enteritidis should be scrutinized.

Bacteriophage Typing↗

PubMed1998

Restriction endonuclease analysis and plasmid profiling of Actinobacillus pleuropneumoniae serotype 7 strains.

Seventeen serotype 7 Actinobacillus pleuropneumoniae strains isolated in New Zealand and A. pleuropneumoniae serotypes 1-12 reference strains were typed by restriction endonuclease analysis of chromosomal DNA and plasmid profiling. All serotype 7 strains produced similar DNA cleavage patterns and were significantly different to other reference serotype strains. Minor differences in the cleavage patterns enabled the 17 serotype 7 strains to be grouped into seven profiles. Plasmids were identified in all but three strains but the banding patterns did not account for the differences in the chromosomal profiles. The study showed that restriction endonuclease analysis and plasmid profiling are useful in epidemiological studies of porcine pleuropneumonia.

Actinobacillus Infections↗

PubMed1978

Conjugal transfer system of plasmid RP4: analysis by transposon 7 insertion.

We have begun an analysis in Escherichia coli of the conjugal transfer functions of the broad-host-range plasmid RP4. We have isolated 19 tra mutants of RP4, generated by insertion of transposon 7, and mapped their insertion sites by restriction endonuclease analysis. These sites fall into two separate regions on either side of the kanamycin resistance determinant. The transfer rates of the mutants range from 10% of that of RP4 to an undetectable level. Spot tests with the P-1 pilus-specific phages PRR1, Pf3, and PR4 and electron microscopic examination for pili have classified the mutants into several phenotypes consistent with their having normal, retracted, or no pili. Analysis of transient plasmid heterozygotes, created by P1 transduction, divided the tra mutants into a minimum of five complementation groups. Some of these groups contain more than one phenotypic class and may represent more than one gene because of the possible polar and deletion effects of Tn7 insertion.

Chromosome Mapping↗

PubMed1990

[Drug resistant plasmids of Pseudomonas pyocyanea in the respiratory tract].

This paper reports the results of the conjugating transmission test, the plasmid elimination test and the plasmid analysis of pseudomonas pyocyanea which were separated from the sputum of the patients with infection of respiratory tract. The result of the conjugating test and the plasmid elimination test of P6P8 strains indicated that the partial drug resistances of two strains are mediated by the drug resistant plasmids. The results of the plasmids analysis in 14 strains of pseudomonas pyocyanea showed that this pyocyanic infection was mainly caused by the bacillus pyocyanea carrying 38 x 10(6) dal plasmids. The technique of plasmid analysis is simple and good repetition. The specificity of this method is analogous to phage typing. This method is worth popular in the epidemic investigation of pathogenic bacteria tracing.

Anti-Bacterial Agents↗

PubMed1996

A rapid mechanical lysing procedure for routine analysis of plasmids from lactobacilli, isolated from sourdoughs.

Variations in cell wall composition of lactobacilli complicate attempts to standardize plasmid detection on the basis of enzymatic lysing procedures. Mechanical disruption of cell walls by a horizontal mixer-mill proved to be more effective, rapid and reproducible. In order to judge the risk of damaging plasmid deoxyribonucleic acids (DNA) in this way, plasmid profiles obtained after enzymatic lysing and mechanical disruption of cell walls were compared. As no differences were detected the mechanical procedure was accepted as a suitable method for routine analysis of plasmid profiles.

Bread↗

PubMed1992

[Comparative plasmid profile analysis of Salmonella typhimurium var. Copenhagen strains from a Salmonella outbreak in hospitalized horses].

From April 1990 through June 1991 clinical salmonellosis and asymptomatic faecal excretion of Salmonella spp. were seen in hospitalized horses at two veterinary hospitals. 76 Salmonella strains from hospitalized horses and 18 strains from horses without any clinical contact were characterized by serotyping and plasmid profile analysis. From April 1990 through January 1991 97.8% of the hospitalized horses were infected with strains of S. typhimurium var. Copenhagen, which were closely related according to their similar plasmid patterns. Other strains of S. typhimurium var. Copenhagen and serotype S. enteritidis were isolated not before February 1991. In the same period various plasmid profile types of S. typhimurium var. Copenhagen and strains of S. typhimurium, S. enteritidis and S. lexington were isolated from horses of the control group. Our results suggest that the high incidence of salmonellosis and latent salmonella infection in hospitalized horses was mainly due to the spread of one particular strain of S. typhimurium var. Copenhagen and that this strain was obviously acquired during hospitalisation.

Animals↗

PubMed1983

Evaluation of isolated cases of salmonellosis by plasmid profile analysis: introduction and transmission of a bacterial clone by precooked roast beef.

In July-August 1981, the number of isolated cases of salmonellosis in New Jersey and Pennsylvania markedly increased. Concurrently, two outbreaks in these states caused by the same Salmonella serotypes were traced to a single brand of precooked roast beef. Plasmid profiles of outbreak and isolated strains were examined, and Salmonella newport isolates from the implicated meat and from cases in the two outbreaks were identified with a unique profile, present in 45% of reported strains from isolated cases in this area during the same period. Review of the food exposure histories in isolated cases demonstrated association between this plasmid profile and consumption of precooked roast beef (P = 0.003). Examination of S newport strains from other regions and intervals indicated that this strain was introduced into these two states sometime after March 1981. Thus, plasmid profile analysis was useful in studying the epidemiology of isolated cases of salmonellosis due to a sensitive common serotype.

Disease Outbreaks↗

PubMed1984

Construction and analysis of plasmids containing the Escherichia coli serB gene.

Plasmid pserB59-1 carries the E. coli serB gene on a 5.2 kb BamHI fragment cloned into the BamHI site of plasmid pBR322. The results of genetic and biochemical experiments established that a functional serB gene is contained in the fragment. The location of the serB gene within the insert was determined by restriction endonuclease analysis of plasmids derived from pserB59-1 that carry the Tn5 element at sites that inactivate the serB gene, and by deletions of segments of the 5.2 kb insert that either inactivate or do not inactivate the serB gene. A 38,000 Mr serB+ polypeptide was detected when plasmid pserB59-1 was used as template in a minicell system, but not when the serB gene was inactivated by insertion of a Tn5 element.

Base Composition↗

PubMed2004

Sequence analysis of plasmid pTS1 isolated from oral spirochetes.

The naturally occurring plasmid pTS1, identified previously in several species of oral spirochetes, has now been completely sequenced. Analysis of the four open reading frames identified on the plasmid suggests the presence of genes involved in replication and mobilization.

Amino Acid Sequence↗

PubMed1990

Plasmid profile analysis and restriction enzyme fingerprinting of Salmonella DO-group strains.

In Sweden Salmonella dublin is the most common serotype within the DO-group isolated from animals. In recent years also salmonella strains belonging to the DO-group but lacking H-antigen have been isolated from cattle in different areas. It was not possible to further differentiate the latter strains by serological methods. However, all Salmonella dublin strains and those strains lacking H-antigen carried a 50 Mdal plasmid exhibiting the same EcoRI and Hind III restriction enzyme digestion pattern. Two of the 26 strains contained an additional 5 Mdal plasmid. Other serotypes within the DO-group investigated, namely Salmonella enteritidis and Salmonella panama, carried plasmids smaller than 50 Mdal. The plasmid profiles and restriction enzyme digestion patterns indicated that the salmonella strains lacking H-antigen were variants of Salmonella dublin. Thus, analysis of plasmid profile and restriction enzyme fingerprinting are useful complements to serological methods in the differentiation of salmonella DO-group strains.

Drug Resistance, Microbial↗

PubMed1995

In vivo stability and discriminatory power of methicillin-resistant Staphylococcus aureus typing by restriction endonuclease analysis of plasmid DNA compared with those of other molecular methods.

We evaluated test discriminatory power and DNA type alterations among methicillin-resistant Staphylococcus aureus strains by testing 199 sequential isolates from 39 patients collected over 30 to 228 days. Isolates were typed by one or three different methods (restriction endonuclease analysis of plasmid DNA [REAP] with or without pulsed-field gel electrophoresis of genomic DNA [PFGE] and immunoblotting [IB]). REAP was highly discriminatory compared with PFGE and IB. However, the initial isolates from 4 of the 39 patients lacked detectable plasmid DNA and could not be typed by REAP. Typing of individual patient isolates showed that a different REAP type was identified only once every 138 days. Among 25 comparisons, seven sequential isolate pairs demonstrating REAP differences were also different by PFGE and IB. This likely represented the presence of more than one strain. Eighteen other pairs with REAP differences were identical or related to one another by PFGE and IB typing, and 17 of these differences were likely caused by a single genetic alteration within the same strain or clone. The rate of PFGE differences explicable by single genetic alterations among sequential isolates identical by REAP was similar to the overall rate for REAP differences in the whole collection. We conclude that REAP and PFGE typing differences explicable by single genetic alterations are relatively infrequent but not rare. These isolates should be examined by alternative typing systems to further support or refute clonality.

Bacterial Typing Techniques↗

PubMed1995

Molecular characterisation of Escherichia coli O157:H7 isolates by pulsed-field gel electrophoresis and plasmid DNA analysis.

Foods of bovine origin have been identified as sources of Escherichia coli O157:H7. Genomic DNA of E. coli O157:H7 isolates from patients (six isolates), food samples (18 isolates from ground beef and six isolates from raw milk) and calf faecal samples (31 isolates) were characterised by pulsed-field gel electrophoresis (PFGE) and plasmid DNA analysis. These isolates originated from different locations in the USA during 1992 and 1993. Twenty-one distinct genomic profiles were generated from the 61 isolates by PFGE after digestion with the endonuclease XbaI. Four genomic profiles were identified among five patient isolates and the remaining patient isolate was not typable. Five different profiles were detected amongst the isolates from ground beef, one of which was associated with 13 ground beef isolates from an outbreak in the Pacific Northwest of the USA in 1993. The PFGE profile of five calf isolates from Washington and Wisconsin was identical to the profile of the ground beef isolates from the outbreak, suggesting that these isolates were related. Similarly, one PFGE profile accounted for three isolates from calf faeces and one from ground beef. Six raw milk isolates from Georgia were indistinguishable both from each other and from one isolate from calf faeces. Fourteen genomic profiles were identified among 31 calf faecal isolates from 18 different herds in 11 states. Only five plasmid profiles were identified among the 61 isolates. PFGE was shown to be a useful typing technique for E. coli O157:H7.

Animals↗

PubMed1987

Subtyping of Legionella pneumophila serogroup 1 isolates by monoclonal antibody and plasmid techniques.

A group of environmental and clinical Legionella pneumophila serogroup 1 isolates was subtyped by monoclonal antibody dot immunoblotting and plasmid analysis. Monoclonal antibody analysis defined seven subtypes within three major groups. Plasmid analysis (including restriction endonuclease digestion) revealed 10 subtypes. By combining plasmid and monoclonal techniques, all 16 strains were shown to be distinct. Plasmid profiles and monoclonal antibody reactivities of selected strains were stable despite serial passage (greater than 100 times). No plasmid-associated antigen was defined by this panel of monoclonal antibodies. The observed dissociation of plasmid profiles and monoclonal antibody reactivity patterns suggests that accurate epidemiologic typing of L. pneumophila serogroup 1 strains will require use of both techniques.

Antibodies, Monoclonal↗