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PubMed1989

[Study of the antimicrobial sensitivity, auxotype, serotype and plasmid analysis of 75 strains of Neisseria gonorrhoeae isolated in Barcelona].

The sensitivity of five antimicrobials (penicillin, cefoxitin, tetracycline, ceftazidime and spectinomycin) was assayed in 75 Neisseria gonorrhoeae strains, isolated from May 1986 to December 1987 in a general hospital in Barcelona. Auxotype, serotype and plasmid analysis studies were also performed on these strains. Twenty-six (34%) penicillinase-producing strains were found. 49% of the non-penicillinase-producing strains showed intermediate resistance to penicillin and 12% were resistant with MIC greater than 1 mg/1. 6% were resistant to tetracycline and cefoxitin, respectively, and all were sensitive to ceftazidime and spectinomycin. All the penicillinase-producing strains had the plasmid Africa and three of them had a diffuser plasmid of 24.5 mD. With regard to the auxotype study, most of the strains were prototrophic (38%) followed by the proline dependent ones (38%). 64% of the penicillinase-producing strains belonged to the serogroup W I, while 77% of the non-penicillinase-producing strains were in the W II/III serogroup.

Adolescent↗
PubMed1995

Polymorphism of rRNA genes and plasmid analysis in the typing of Salmonella enterica serovar enteritidis from a Spanish health area.

A molecular epidemiological study of Salmonella enterica serovar Enteritidis (S. enteritidis) in a Spanish Health Area was carried out using two genotypic methods; polymorphism of rRNA genes (ribotyping) and plasmid analysis. The series included 100 isolates randomly selected from among those collected over the period 1984-92 (50 from sporadic episodes and 50 from 10 outbreaks). Ribotyping using HindIII, SmaI, and EcoRI showed a notable degree of homogeneity within Enteritidis serovar, it being able to discriminate only 1, 2, and 3 ribotypes, respectively. Such data suggest that there are a limited number of types or clonal lines, of which only one was widely disseminated within the health area under study (including 90% of isolates from sporadic episodes and all of those from outbreaks). Twelve plasmid profiles were differentiated. Sixty isolates carried only the virulence plasmid of 36 Md; whereas 24 isolates, grouped into 10 profiles, carried the 36 Md together with other plasmids; 7 isolates carried only other plasmids and 9 isolates were plasmid free. The combination of results from both methods revealed 15 subtypes, 9 of these including two or more isolates, one subtype being endemic and predominant (54% of the sporadic isolates and isolates from 6 outbreaks).

Bacterial Typing Techniques↗
PubMedDate not supplied

Shigellosis in day care centers: use of plasmid analysis to assess control measures.

Spread of shigellosis within day care centers is difficult to control, and illness may spread into the community. We investigated two sequential outbreaks of diarrhea caused by multiply resistant Shigella sonnei in children at day care centers in 2 neighboring counties in Florida. We used plasmid analysis to identify a single epidemic strain causing both outbreaks and to assess the control measures used. One of the control measures, keeping ill children from attending the center, did not prevent spread of illness in the community.

Child Day Care Centers↗
PubMed1986

Plasmid analysis of simultaneous nosocomial outbreaks of methicillin-resistant Staphylococcus aureus.

A large outbreak of infections caused by methicillin and aminoglycoside resistant Staphylococcus aureus provided the opportunity to evaluate mechanisms of resistance and compare the usefulness of typing systems. Between January 1979 and December 1980, 63 patients developed infections with S aureus resistant to multiple antibiotics, including methicillin and tobramycin. All isolates had an identical antibiogram and were phage type 47/54/75/77/83A. Beginning in January 1981, a superimposed outbreak caused by S aureus of the same phage type but with a resistance pattern now including gentamicin occurred. The two strains contained different aminoglycoside inactivating enzymes. The initial strain contained a single plasmid of 21.5 mDa molecular weight, whereas the subsequent strain which had acquired gentamicin resistance contained this plasmid plus a heavier one of 33 mDa. Plasmid analysis complements the analysis of antibiograms and phage types and aids in defining epidemiologic patterns of transmission.

Bacteriophage Typing↗
PubMed1992

Plasmid analysis in PPNG strains isolated in Italy.

Italian reports of PPNG strains are rare. In this paper we report the plasmidial characteristics of two strains: one harbouring 3.2 and 2.6 Mdal plasmids and the other 24.5, 4.5 and 2.6 Mdal plasmids. Restriction analysis was carried out on plasmids transferred to E. coli by transformation for "Africa" and conjugation for "Asia" plasmid.

DNA, Bacterial↗
PubMedDate not supplied

Grown gall plant tumors of abnormal morphology, induced by Agrobacterium tumefaciens carrying mutated octopine Ti plasmids; analysis of T-DNA functions.

Ti plasmid mutants derived from Agrobacterium tumefaciens strain Ach5 that induce tumors of abnormal morphology have been analyzed. On tobacco, A. tumefaciens mutant strain LBA4060 induces tumors that specifically give rise to shoots. Shoots continue to grow from in vitro cultured bacteria-free tumor tissue derived from such tumors. The mutant character is shown to be correlated with the insertion of an A. tumefaciens IS element, IS60, into the left arm of the T-region of the octopine Ti plasmid. Evidence is presented showing that IS60 is transferred into the plant cell DNA as part of the T-DNA. A second Ti plasmid insertion mutant A. tumefaciens strain LBA4210, with a Tn904 transposon in the center of the T-region, induces tumors that specifically exhibit a root development on tobacco plants. T-DNA has been detected in sterile amorphous crown-gall tissue derived from these tumors. The transposon Tn904 insertion was shown to result a changed "core" T-DNA. Abnormal tumor morphologies induced by these mutant strains have been observed also on Kalanchoë stems. On tomato plants the mutants induce small unorganized tumors while on Nicotiana rustica unorganized tumors, nearly equal in size to those caused by the wild-type strain have been induced. LBA4060 was shown to be avirulent on Kalanchoë leaves and LBA4210 was weakly virulent. Infection of Kalanchoë leaves or tomato plants with a mixture of separately grown cultures of both mutants resulted in the formation of more or less normal tumors. The exposure of a tomato plant to naphthalene acetic acid (NAA), a synthetic auxin, during development of tumors induced by LBA4060 stimulated tumor formation. Tumor growth induced by LBA4210 was found to be stimulated by kinetin.

Cloning, Molecular↗
PubMed1987

Plasmid analysis as an epidemiological tool in neurosurgical infections with coagulase-negative staphylococci.

Coagulase-negative staphylococci isolated from blood or spinal fluid during a period of 1 year in a department of neurosurgery, were analysed by biotyping, antibiotic resistance pattern and plasmid profiles. Altogether 41 isolates from 19 patients were studied. About 90% of the isolates were Staphylococcus epidermidis. The antibiotic resistance pattern seemed to be closely related to antibiotic usage in the unit. Most common was resistance to penicillin (63%), trimethoprim-sulphamethoxazole (49%) and cloxacillin (39%) while resistance to gentamicin was seen in only one strain. In several cases species and antibiograms were identical in isolates from different patients. Plasmid pattern analysis could then be used for identification of different strains. In one instance, plasmid pattern and restriction enzyme analysis confirmed that two patients probably were infected by the same strain.

Anti-Bacterial Agents↗
PubMed1998

Genotypic characterization of Salmonella enteritidis phage types by plasmid analysis, ribotyping, and pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) was used to resolve XbaI and SpeI macrorestriction fragments from 60 defined phage type (PT) reference strains of Salmonella enteritidis. The level of discrimination was compared to that afforded by plasmid profile analysis and ribotyping. Twenty-eight distinct XbaI pulsed-field profiles (PFPs) were observed, although a single type, PFP X1, predominated. Absence of the 57-kb spv-associated fragment was observed for three PT reference strains, and the profile was designated PFP X1A. The XbaI macrorestriction profiles of a further four PT reference strains were altered by the presence of plasmid-associated bands. Twenty-six SpeI-generated PFPs (plus one subtype) were observed for the same strains. No SpeI fragment corresponding to the 38-MDa serovar-specific plasmid was detected. The distribution of XbaI and SpeI profiles did not always correspond, producing a total of 32 combined PFPs for the 60 PT reference strains. This compared with a total of 18 different plasmid profiles and three PvuII ribotypes generated by the same strains. The results of this study indicate that PFGE may offer an improved level of discrimination over other genotypic typing methods for the epidemiological typing of S. enteritidis.

Bacterial Typing Techniques↗
PubMedDate not supplied

Molecular typing of multi-drug resistant Shigella dysenteriae type 1 by plasmid analysis and pulsed-field gel electrophoresis.

Recently, an outbreak of dysentery due to multi-drug resistant Shigella dysenteriae type 1 strains was reported along the coastal area of Kenya and shortly thereafter another outbreak appeared in the outskirts of Nairobi. We analysed 22 multi-drug resistant S. Dysenteriae type 1 strains isolated from cases in the latter outbreak using plasmid deoxyribonucleic acid (DNA) profiles and pulse-field gel electrophoresis of genomic DNA. All isolates were resistant to commonly available drugs including ampicillin, trimethoprim, sulphamethoxazole, chloramphenicol, tetracycline and streptomycin with minimum inhibitory concentrations > 64 micrograms/mL, but were fully sensitive to gentamicin. Only 2 strains were resistant to nalidixic acid. Analysis of plasmid DNA and genomic DNA revealed that all 22 strains were clonally related. It is likely that the present outbreak was related to that on the coast, as suggested by the similarity in drug susceptibility data. The drug susceptibility and molecular epidemiological data provide a useful baseline for future monitoring of epidemic and endemic S. dysenteriae activity in East Africa.

Animals↗
PubMed1990

Plasmid analysis of clinical isolates of Bacteroides fragilis group strains.

Cryptic plasmids were isolated from 26 of 52 Bacteroides fragilis group strains derived from severe infections. Four strains harboured two plasmids, one three plasmids and one five plasmids with different molecular weights. The same molecular weight plasmid (3.7 Md) was isolated from 17 of the 26 plasmid-containing B. fragilis group strains. No correlation was found between plasmid-harbouring and resistance against ten antibiotics and different heavy metal ions. No curing of the strains from the plasmids was achieved with ethidium bromide and acridine orange.

Anti-Bacterial Agents↗
PubMed1995

Plasmid analysis of Australian strains of Salmonella enteritidis.

Using an in-well lysis technique, 73 Australian strains of Salmonella enteritidis were shown to possess a large plasmid, similar in size to that possessed by a reference phage type 4 strain. Restriction analysis of the large plasmid from nine strains using EcoRI, HindIII and PstI suggested that these plasmids are similar to or the same as the 38 MDa plasmid described in strains of this species from other parts of the world.

Australia↗
PubMed1983

Investigation of nosocomial infections by plasmid analysis.

Molecular biological techniques, including agarose gel electrophoresis, restriction endonuclease analysis and DNA-DNA hybridization (Southern blotting and heteroduplex analysis) are being applied very successfully to the investigation of nosocomial infections. 'Plasmid fingerprinting' by electrophoresis, with or without restriction endonuclease analysis, can be used to identify epidemic strains of bacteria and 'epidemic plasmids' which have spread through several different bacterial species. This technique is rapid and inexpensive, and can be applied to drug-sensitive as well as resistant strains. This approach is especially useful for investigation of organisms for which no standard typing system is available. DNA-DNA hybridization techniques can be used to study the evolution of plasmids in the hospital environment, and to demonstrate the presence and spread of translocatable DNA sequences (transposons) carrying drug resistance determinants from plasmid to plasmid within a bacterial cell.

Cross Infection↗
PubMed1989

Plasmid analysis in pink facultative methylotrophic bacteria using a modified acetone-alkaline hydrolysis method.

Routine screening of indigenous and recombinant plasmids in pink facultative methylotrophic bacteria has been difficult, time-consuming, and yields variable results. We report a modified alkaline hydrolysis method for rapid plasmid isolation from these organisms that reproducibly results in good yields of closed circular plasmid DNA which can be readily digested with restriction enzymes. This method greatly facilitates direct screening of indigenous and introduced recombinant plasmids in the methylotrophic host strain. We have confirmed earlier findings that the original NCIB wild-type strain of Methylobacterium sp. strain AM1 (NCIB 9133) contains three cryptic plasmids. However, sizing of these plasmids by comparison to standards and by restriction fragment analysis suggests that they are larger than previously reported. We have designated these plasmids pAM1-1 (65 kb), pAM1-2 (40 kb) and pAM1-3 (33 kb). We have also shown that a rifamycin-resistant strain of Methylobacterium sp. strain AM1 used routinely in our laboratory lacks pAM1-2, although no phenotype has been associated with its loss. Finally, we have shown that another pink facultative methylotroph, Methylobacterium isolate (#YK1), contains three cryptic plasmids of approximately 43, 37 and 22 kb, respectively.

Acetone↗
PubMed1993

Molecular subtyping by genome and plasmid analysis of Campylobacter jejuni serogroups O1 and O2 (Penner) from sporadic and outbreak cases of human diarrhoea.

Ribosomal RNA gene patterns, randomly amplified polymorphic genomic DNA (RAPD) profiles and plasmid profiles were used to discriminate between 28 strains of Campylobacter jejuni serogroups O1 and O2 (Penner). Most isolates were biotype I (Lior). The strains were representative isolates from a UK school outbreak of enteritis (7 cases) and from 21 sporadic human cases of enteritis in 4 countries. The molecular techniques discriminated to various degrees between strains in each of the serogroups. The outbreak strains were homogeneous in most molecular features but a variety of types was detected amongst the isolates from the sporadic cases. Five groups of two or more strains with identical ribopatterns were identified and within each, strains from different patients were homogenous with respect to serogroup. RAPD profile typing based on numerical analysis generally matched ribotyping. Plasmid profiling overall gave least discrimination but was useful in separating some strains similar in other features. We concluded that optimal discrimination of C. jejuni could best be achieved using a combination of phenotypic and genotypic properties. Hae III ribotyping was the single most discriminatory and reproducible technique investigated. Several strains of C. jejuni from sporadic infections had similar molecular profiles which have potential for general typing purposes.

Bacterial Typing Techniques↗
PubMed1989

Plasmid analysis and antimicrobial susceptibilities of Peptostreptococcus species.

There are no published methods on plasmid isolation from Peptostreptococcus spp., therefore two methods of plasmid isolation from this genera were analysed: the boiling and alkaline-SDS methods. Plasmid DNA was not recovered by the boiling method, however, with the alkaline-SDS method, cryptic plasmid DNA was detected in two P. asaccharolyticus and one P. magnus strains. To achieve optimum lysis, Peptostreptococcus cells were treated with lysozyme (2 mg/ml) for 15 min. at 37 degrees C followed by proteinase K (0.2 mg/ml) for 1 h at 37 degrees C. In addition we report, the occurrence of clindamycin or metronidazole-resistant peptostreptococci, but these phenotypes were not correlated with plasmid carriage.

DNA, Bacterial↗
PubMed1996

Plasmid analysis of 26 staphylococcal species by a rapid microscale technique.

OBJECTIVE: Evaluate a plasmid typing technique for a diverse group of staphylococci. DESIGN: In vitro testing on known isolates. SETTING: University hospital. INTERVENTION: The plasmid content of 195 isolates representing 26 staphylococcal species was analyzed by agarose gel electrophoresis following lysostaphin-alkaline-SDS lysis, with and without acetone treatment. RESULTS: Isolates yielded plasmid profiles with 0 to 7 extra-chromosomal bands (median, 1; 1.5); 171 (88%) had a profile with at least 1 band. Species with more than one isolate available for testing showed considerable diversity of plasmid profiles, except for Staphylococcus haemolyticus. CONCLUSIONS: The ease of the procedure and the diversity of plasmid profiles within each species examined suggests that plasmid profiling is an accessible and useful epidemiologic tool applicable to most staphylococcal species by epidemiologic units or clinical laboratories.

Bacterial Typing Techniques↗
PubMed1988

Plasmid analysis of Shigella dysenteriae type 1 isolates obtained from widely scattered geographical locations.

Plasmid profiles and antimicrobial susceptibility patterns of 343 strains of Shigella dysenteriae type 1, obtained from 18 different geographical locations, were analyzed. Three plasmids, with molecular sizes of 140, 6, and 2 megadaltons (MDa), were present in 94, 98, and 96%, respectively, of the 343 strains isolated during either epidemic or nonepidemic periods from 1965 to 1987. In addition to these plasmids, 83% of the strains harbored a 4-MDa plasmid and 25% harbored a 20-MDa plasmid. Various plasmid profiles were observed in which the 140-, 6-, and 2-MDa plasmids occurred commonly, irrespective of the place of isolation and drug resistance pattern of the strains. Certain profiles showed significant association with drug resistance patterns. These findings suggest that three plasmids, of molecular sizes 140, 6, and 2 MDa, are unique to S. dysenteriae type 1 strains and may indicate the global spread of a pathogenic bacterial clone. Additionally, these core plasmids, plus plasmids of various other sizes, could be used to identify emerging subclones which are causing both epidemic and sporadic disease. Thus, plasmid profiles of S. dysenteriae type 1 strains can be used to monitor possible pandemic strains as well as individual epidemic strains.

Drug Resistance, Microbial↗