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PubMed1983

Analysis of plasmids in nosocomial strains of multiple-antibiotic-resistant Staphylococcus aureus.

Nosocomial infections caused by Staphylococcus aureus strains resistant to methicillin and multiple antibiotics have reached epidemic proportions in Melbourne, Australia, over the past 5 years. Plasmid analysis of representative clinical isolates demonstrated the presence of three classes of plasmid DNA in most strains. Resistance to gentamicin, kanamycin, and tobramycin was usually mediated by an 18-megadalton plasmid but could also be encoded by a related 22-megadalton plasmid. Two distinguishable plasmids of 3 megadaltons each endowed resistance to chloramphenicol, and the third class consisted of small plasmids, each approximately 1 megadalton in size, with no attributable function. An extensive array of resistance determinants, including some which have usually been associated with a plasmid locus, were found to exist on the chromosome. Evidence that resistance to gentamicin, kanamycin, and tobramycin is chromosomally encoded in some clinical isolates suggests that this determinant may have undergone genetic translocation onto the staphylococcal chromosome.

Aminoglycosides↗

PubMed1995

Correlation of six methods for typing nosocomial isolates of Acinetobacter baumannii.

A comparative study of biotyping, antimicrobial susceptibility, whole-cell protein analysis, plasmid analysis, pulsed-field gel electrophoresis of chromosomal DNA and polymerase chain reaction with arbitrary primers of Acinetobacter baumannii isolates from three large hospitals was performed to determine the best markers for epidemiological purposes. Ninety-two isolates were included: 38 belonged to a previously described outbreak and 54 were randomly selected from sporadic cases of infection. Biotyping, whole-cell protein and plasmid analysis were the least discriminatory methods, whereas antimicrobial susceptibility and polymerase chain reaction with arbitrary primers showed moderate discriminatory power. Typing based on pulsed-field gel electrophoresis of chromosomal DNA appeared to be the best discriminatory method (discrimination index of 0.9623). The addition of polymerase chain reaction with arbitrary primers or antimicrobial susceptibility to pulsed-field gel electrophoresis of chromosomal DNA did not further increase the discriminatory power.

Acinetobacter↗

PubMed1992

Molecular typing of ampicillin-resistant, non-beta-lactamase-producing Enterococcus faecium isolates from diverse geographic areas.

Molecular typing methods were compared by using 66 ampicillin-resistant, non-beta-lactamase-producing Enterococcus faecium clinical isolates from diverse geographic areas. Whole-plasmid analysis, restriction enzyme analysis of plasmid DNA with EcoRI and HindIII, and contour-clamped homogeneous electric field electrophoresis with digestion by SmaI and ApaI were performed on all isolates. Whole-plasmid analysis identified 47 different groups. Restriction enzyme analysis of plasmid DNA identified 50 groups when EcoRI was used and 51 groups when HindIII was used. Results with EcoRI and HindIII differed in 9 of 66 isolates. Grouping results with whole-plasmid analysis differed from results of restriction enzyme analysis of plasmid DNA (combining EcoRI and HindIII) in 20 of 66 isolates. Contour-clamped homogeneous electric field electrophoresis identified 46 groups when SmaI was used and 44 groups when ApaI was used. Results with SmaI and ApaI differed in 3 of 66 isolates. Grouping results with contour-clamped homogeneous electric field electrophoresis (combining SmaI and ApaI) differed from results of restriction enzyme analysis of plasmid DNA (combining EcoRI and HindIII) in 17 of 66 isolates. The combined use of whole-plasmid analysis, restriction enzyme analysis of plasmid DNA with two enzymes, and contour-clamped homogeneous electric field electrophoresis with two restriction enzymes should be considered when E. faecium is typed for epidemiologic investigation.

Ampicillin Resistance↗

PubMedDate not supplied

[Cloning of bacteriophage T5 DNA fragments in the plasmid pBR322. Analysis of recombinant plasmids by the method of bonding with RNA-polymerase from Escherichia coli on nitrocellulose filters].

DNA of bacteriophage T5 was hydrolyzed with restriction endonucleases HindIII and BamHI, and subjected to the combined hydrolysis with BamHI+EcoRI and BamHI+ +HindIII. Fragments obtained were cloned in the plasmid pBR322. About 17% of T5 genome were recovered in recombinant plasmids. Cloned fragments were localized on the physical map of the phage by restriction analysis and Southern hybridization. With the aim of direct cloning of T5 promoters, PstI/HindIII fragments were inserted into pBR322 followed by selection of recombinants on ApsTCr phenotype. Binding of BsuRI and AluI fragments of hybrid plasmids with E. coli RNA polymerase was studied by nitrocellulose filter assay. The fragments, which were capable to form heparin resistant complexes were identified.

Base Sequence↗

PubMed1994

Usefulness of molecular epidemiology for outbreak investigations.

We conducted a retrospective review of nosocomial outbreak investigations conducted by the Hospital Infections Program, Centers for Disease Control and Prevention, from January 1991 through March 1994. Selected outbreaks have demonstrated the utility of molecular methods such as plasmid analysis, plasmid restriction endonuclease analysis, ribotyping, restriction fragment polymorphism, pulsed-field gel electrophoresis, and polymerase chain reaction in confirming the clonality of the outbreak and in confirming the source of the outbreak implicated in the epidemiologic investigation. These data show that molecular typing of isolates is particularly useful when combined with epidemiologic investigations of nosocomial outbreaks.

Bacterial Typing Techniques↗

PubMed2004

Characterization of Salmonella enterica serovar typhimurium from marine environments in coastal waters of Galicia (Spain).

Twenty-three Salmonella enterica serovar Typhimurium isolates from marine environments were characterized by phage typing, pulsed-field gel electrophoresis (PFGE) analysis, plasmid analysis, and antibiotic resistance, and the distribution of the different types in the coastal waters were subsequently analyzed. Five phage types were identified among the isolates (PT41, PT135, PT99, DT104, and DT193). PT135 isolates were exclusively detected during the winter months from 1998 to 2000, whereas DT104 and PT41 isolates were detected exclusively in the summer months from 2000 to 2002. XbaI PFGE analysis revealed 9 PFGE types, and plasmid profiling identified 8 plasmid types (with 1 to 6 plasmids) among the isolates. Only three isolates presented multidrug resistance to antibiotics. Two DT104 isolates were resistant to 8 and 7 antibiotics (profiles ACCeFNaSSuT and ACeFNeSSuT), whereas a PT193 isolate presented resistance to 6 antibiotics (profile ACFSSu). In addition, four PT41 isolates were resistant to a single antibiotic. The detection of multidrug-resistant phage types DT104 and DT193 in shellfish emphasizes the importance of monitoring the presence of Salmonella in routine surveillance of live bivalve molluscs.

Drug Resistance, Bacterial↗

PubMed2006

Molecular characterization and antibiotic resistance profiling of Salmonella isolated from retail Turkey meat products.

Contaminated poultry meat has been identified as one of the principal foodborne sources of Salmonella. Molecular characterization of Salmonella is important in addressing methods to control this pathogen. Seventy-four retail turkey meat samples were collected from various stores in Fargo, North Dakota in the fall of 2003. Salmonella was recovered from 30 samples using the standard conventional culture method (FSIS, USDA). Isolated Salmonella were characterized by serotyping, pulsed-field gel electrophoresis (PFGE) analysis, plasmid analysis, and antibiotic resistance profiling. Five serotypes were identified among the isolates: Newport (n = 12), Hadar (n = 8), Heidelberg (n = 7), 4,12:nonmotile (n = 2), and Reading (n = 1). XbaI PFGE analysis revealed 13 PFGE types and succeeded in grouping the isolates according to their serotypes. Plasmid profiling identified 5 plasmid types (with 1 or 2 plasmids) among eleven isolates that harbored plasmids. Seventeen isolates were resistant to antibiotics. The Heidelberg serotype showed resistance to multiple antibiotics: 1 isolate had resistance to gentamicin, sulfamethoxazole, and streptomycin, and 6 isolates had resistance to tetracycline, gentamycin, sulfamethoxazole, kanamycin, and streptomycin. The Hadar serotype isolates were resistant to 2 or 3 antibiotics: tetracycline and streptomycin (1 isolate); tetracycline and kanamycin (1 isolate); and tetracycline, kanamycin, and streptomycin (6 isolates). The 4,12:nonmotile serotype isolates showed resistance to tetracycline only. The Newport and the Reading serotypes were susceptible to all 16 of the antimicrobials tested.

Animals↗

PubMed2005

Molecular typing of Klebsiella pneumoniae isolates from public hospitals in Recife, Brazil.

Thirty nosocomial isolates of Klebsiella pneumoniae, collected from three public hospitals in Recife, Brazil, between 1999 and 2000, were analysed in order to determine their epidemiological relatedness and genetic characteristics. The isolates were characterised by biotyping, antibiotyping, protein analysis, plasmid profile and random amplified polymorphic DNA (RAPD). The majority of the clinical isolates were resistant to multiple antibiotics, in particular to beta-lactams, and 30% were found to be ESBLs producers. RAPD proved to be the most effective technique in discriminating unrelated K. pneumoniae isolates. It was confirmed by the highly genetic similarity found among related isolates from an hospital outbreak. We conclude that K. pneumoniae infections in Recife has been caused by a variety of bacterial genotypes. This is the first report that revealed the subtypes of K. pneumoniae in Brazil by plasmid analysis and RAPD.

Anti-Bacterial Agents↗

PubMed1994

Epidemiological markers of Acinetobacter baumannii clinical isolates from a spinal cord injury unit.

During a period of 28 months, 114 isolates of Acinetobacter baumannii obtained from urine samples of 57 patients, were recovered in a Spinal Cord Unit; an unusual increase in the number of A. baumannii isolates was observed between February 1991 and January 1992. Six different typing methods [biotyping, antimicrobial susceptibility, whole cell and cell-envelope protein analysis, plasmid analysis and chromosomal DNA analysis by pulsed-field gel electrophoresis (PFGE)] were used to study the isolates to establish any potential relationships among them. Chromosomal DNA analysis by digestion with ApaI and separation of the fragments by PFGE was the most powerful tool to determine the relatedness of isolates. The results suggest that the isolates from 1991 and 1992 may have originated from strains present in 1990 that subsequently acquired resistance to amikacin and tobramycin during the epidemic.

Acinetobacter↗

PubMed1988

Identification and cloning of a plasmid-encoded erythromycin resistance determinant from Lactobacillus reuteri.

Plasmid analysis, plasmid curing, cloning, and hybridization experiments were used to study four Lactobacillus reuteri strains showing high resistance to erythromycin. Plasmid curing with acriflavine resulted in a loss of erythromycin resistance in a frequency of 1-10%. For three of the strains this was accompanied by a loss of a 6.9-MDa plasmid, which was shown to be identical for the different strains and designated pLUL631. The erythromycin (erm) gene was located on a 5.5-MDa plasmid in the fourth strain. A restriction map of pLUL631 was constructed and the location of the erm gene on the plasmid was identified by cloning in Escherichia coli. By using a Streptococcus lactis-E. coli shuttle vector, the erm gene was also transformed to S. lactis and expressed. The erm gene from L. reuteri was shown to be related to the erm gene from pIP501 (Streptococcus agalactiae) by DNA-DNA hybridization.

Cloning, Molecular↗

PubMed1991

Gentamicin-resistant Salmonella typhimurium phage type 204c: molecular studies and strain diversity in a putative bovine outbreak.

As part of the investigation of a putative bovine outbreak, 13 isolates of Salmonella typhimurium phage type 204c were subjected to plasmid analysis. Plasmid profiles suggested that several distinct strains were involved and these observations were supported by minor variations in antibiotic resistance pattern. Restriction enzyme fingerprinting and conjugational segregation of the plasmids confirmed these findings. Although 12 of the 13 isolates were resistant to gentamicin, resistance was conferred by 4 distinct plasmids; 3 of these belonged to Inc I and were distantly related on the basis of restriction fingerprints and the fourth was a resistant derivative of the 60 MDa S. typhimurium serotype-specific plasmid. The molecular evidence refuted the hypothesis that geographical and temporal clustering of these gentamicin-resistant isolates could be explained on the basis of a single epidmiological episode.

Animals↗

PubMed1989

Spreading of streptomycin antibiotic resistance gene in Escherichia coli plasmids demonstrated by Southern blot analysis.

Plasmids from E. coli strains of 38 donors were transconjugated to common recipient SY663 Escherichia coli K12. The restriction patterns of the isolated plasmids were highly heterogenous. However, the streptomycin (Sm) resistance genes of the plasmids were identical or closely homologous in 29 of the 33 plasmids conferring Sm resistance. These data were based on Southern blot analysis, using the Sm resistance gene (encoding aminoglycoside phosphoryl transferase) as probe cut out from pBP1 plasmid. Our data suggest an extensive spreading of streptomycin resistance gene of this type.

Blotting, Southern↗

PubMed2005

Comparison of antibiotic susceptibility tests, plasmid profiles and restriction enzyme analysis of plasmid DNA of methicillin susceptible and resistant-Staphylococcus aureus strains isolated from intensive care units.

OBJECTIVE: To differentiate methicillin-resistant Staphylococcus aureus (S. aureus) (MRSA) and methicillin-sensitive S. aureus (MSSA) strains and detect the source of epidemic strains and prevent their access to patients. METHODS: All the procedures were carried out in the Department of Microbiology, Medical Faculty Hospital, Dokuz Eylul University, Izmir, Turkey from 1996-1998, and antibiotic susceptibility tests continued in the laboratory of King Fahad Hospital, Al-Baha, Kingdom of Saudi Arabia (KSA), from 2001-2004. A total of 81 S. aureus strains (71 MRSA, 10 MSSA) from Turkey were isolated from different sites of patients in Intensive Care Unit's (ICU's), evaluated by plasmid profile, Restriction Endonuclease Analysis of Plasmids (REAP), and antibiotic sensitivity tests. A total of 117 S. aureus strains (24 MRSA, 93 MSSA) from KSA were isolated from different sites of patients in ICU's, evaluated by antibiotic sensitivity tests as recommended by National Committee for Clinical Laboratory Standards (NCCLS). RESULTS: Seventy-one MRSA from Turkey were divided into 13 groups by antibiotic sensitivity tests and into 4 groups by plasmid profiles, in which 3rd and 4th groups subdivided into 2 subgroups, and into 5 groups by REAP. The 1st, 2nd, 3rd and 5th groups were subdivided into 2 subgroups. Ten MSSA were divided into 4 groups by antibiotic sensitivity tests, 3 in plasmid profiles and 2 in REAP tests. Twenty-four MRSA strains from KSA were divided into 9 groups by antibiotic sensitivity tests while 93 MSSA strains were divided into 7 groups. CONCLUSION: In respect to epidemiological survey, plasmids profiles and REAP seems to discriminate more respect to antibiotic sensitivity tests but at the same time neither of them were 100% accurately differential. According to the plasmid profile of the 3rd MSSA (Turkey) group, a multi-drug resistance by antibiotic susceptibility tests were noticed and showed the same plasmid profile in MRSA first subgroup of the 3rd group, but the same groups were different in REAP tests. In order to distinguish the discriminatory power of the strains, where REAP is better than plasmid profile and antibiotic sensitivity tests, we may formulate the statement into the following; REAP > plasmid profile > antibiotic sensitivity tests. For typing and gathering of epidemiological data, it is suggested that all 3 methods should be employed in clinical laboratories as they are cheap, practical and easily interpreted.

DNA, Bacterial↗

PubMed1979

Structure and function of plasmid ColE1 and related plasmids.

Analysis of plasmid ColE1, its naturally occurring relatives ColK and CloDF13, and a wide range of ColE1 derivatives containing either insertions or deletions of genetic material has allowed localization on the ColE1 genome of DNA sequences responsible for colicin E1 synthesis, immunity to colicin killing, conjugal mobility and incompatibility. We have examined incompatibility between pairs of ColE1 derivatives ranging in size from 2.6 to 13.8 Md. Though all the plasmids tested exerted ColE1 incompatibility, a definite pattern was observed regarding the dominant plasmid in any pair tested (i.e. the plasmid that displaces the other from a heterozygote). Usually the larger plasmid is displaced. We conclude that loci for incompatibility reside within 0.7 kb of the ColE1 replication region. A model is presented to explain both the incompatibility data and the observation that the fraction of total DNA occurring as ColE1-like plasmid in a cell is approximately constant. Transposons Tn1 and Tn3 (3.2 Md; Apr and approximately 85% homologous), Tn501 (5.5 Md; Hgr), and Tn7 (9.3 Md; Tpr Smr) can all be transposed into ColE1. Though all have closely related. Tn501 and Tn7 do not complement transposition of Tn3 transposition defective deletions. A Tn3-specified 19,000 dalton protein is absent in one particular class of transposition-defective deletion.

Bacterial Proteins↗

PubMed1991

Molecular epidemiology of Shigella infections: plasmid profiles, serotype correlation, and restriction endonuclease analysis.

Plasmid isolation was used to refine the epidemiologic analysis for 168 shigellosis cases in Pima County, Ariz. Plasmids of less than 20 kb were used for comparison of plasmid profiles. Plasmid patterns for each species were distinct. A total of 57 of 74 (77%) Shigella flexneri strains could be placed into seven plasmid patterns, 70 of 79 (89%) Shigella sonnei strains could be placed into seven patterns, 12 Shigella boydii strains could be placed into six patterns, and each of 3 Shigella dysenteriae strains differed. There was a correlation between plasmid patterns and serotypes for S. flexneri, and multiple plasmid patterns were found in serotypes 1, 2, and 6, offering a refinement beyond serotyping. In previous studies we found an association between Mexican travel and an S. sonnei 5.1-kb plasmid. When this plasmid was used as a probe, strong homology was seen with numerous small plasmids in all Shigella species: restriction endonuclease analysis revealed a 1.1-kb AvaI-AvaII fragment common to various plasmids of S. sonnei. S. flexneri, and S. boydii independent of species. Of 34 Pima County Shigella isolates from the mid-1970s. 8 showed plasmid patterns similar to those of the recent isolates. Some plasmids from S. sonnei, S. flexneri, and S. boydii strains isolated in the 1970s also contained the AvaI-AvaII fragment. The conservation of this specific fragment in our population for more than 12 years suggests that it may contain genes important in virulence or survival.

Arizona↗

PubMed1983

Retrospective analysis of plasmid patterns in a study of burn unit outbreaks of infection due to Enterobacter cloacae.

To evaluate the usefulness of plasmid pattern analysis as an epidemiologic tool and to determine the mechanism of resistance to silver sulfadiazine and mafenide acetate, silver sulfadiazine-resistant, mafenide acetate-resistant Enterobacter cloacae isolates from a 1976 outbreak of infections in a burn unit (group I) were analyzed retrospectively. These strains were compared with various E cloacae isolates (groups II-V), including E cloacae isolates from a more recent (1982) burn unit outbreak (group V). Agarose gel electrophoresis (AGE) of DNA isolated from "epidemic" strains of E cloacae revealed a uniform pattern of four plasmid DNA bands, ranging in molecular size from 66 to 2 megadaltons. No such plasmid pattern was seen in silver sulfadiazine-, mafenide acetate-susceptible isolates of E cloacae in groups II, III, and V. Analysis of non-E cloacae coisolates suggested that silver sulfadiazine resistance was associated with the 55-megadalton plasmid. Plasmid pattern analysis easily separated "epidemic" E cloacae isolated in 1976 from those isolated in 1982.

Burns↗

PubMed1999

Quantitative analysis of plasmid forms by agarose and capillary gel electrophoresis.

Plasmids may appear in different forms: circular with different degrees of coiling, partially cleaved or linear, and multimeric as concatamers or catenates. Capillary gel electrophoresis (CGE) of plasmid samples allows the determination of plasmid form distribution. Monomeric and dimeric plasmid DNA forms were separated by both CGE and agarose gel electrophoresis (AGE). The pattern of isoform bands from AGE was compared to the corresponding peak pattern from CGE, and differences in the relative mobility of the plasmid forms between the two methods were found. The comparison of AGE and CGE allows the assignment of AGE bands to CGE peaks. Additionally, the different isoforms can now be quantified by CGE. Routine plasmid form analysis by CGE may be automated, allowing easy, fast, and highly reliable quantification. CGE also offers high resolution and the amount of DNA required is very low. Therefore this method is very useful for the analysis of therapeutics based on plasmid DNA during their production, isolation, and formulation.

Chromatography, High Pressure Liquid↗

PubMed1991

Analysis of plasmid pattern in paediatric intensive care unit outbreaks of nosocomial infection due to Enterobacter cloacae.

Sequential outbreaks of nosocomial infection due to multiply-resistant Enterobacter cloacae occurred in September 1987, and between December 1988 and January 1989, in a paediatric intensive care unit. A total of eight neonates were affected and most had received ventilatory support. Initially, we were unable to determine whether the two outbreaks were caused by the same strain of E. cloacae. After applying plasmid profile analysis to identify epidemic strains, we established that the strain from the first outbreak was different from the second outbreak strain, as each had its own plasmid pattern. During the second outbreak, an environmental bacteriological survey was carried out. We found that the distilled water containers were contaminated with E. cloacae which had the same plasmid profile. After changing the distilled water containers and by reinforcement of aseptic techniques, the nosocomial outbreak was terminated.

Cross Infection↗