Restriction Endonuclease Analysis Of Plasmid DNA Research Articles

A nosocomial outbreak due to non-encapsulated Haemophilus influenzae: analysis of plasmids coding for antibiotic resistance

An outbreak of infections with non-encapsulated Haemophilus influenzae, resistant to ampicillin, chloramphenicol, sulphonamide and tetracycline involved 13 elderly patients and three nurses on acute admission and care of the elderly wards. Thirty-two isolates were found to be indistinguishable on analysis of biotype, antibiogram, serotype and major outer membrane proteins (MOMP). Plasmids could not be identified in the original isolates but after mating with a Rec A H. influenzae recipient, the resultant transconjugates were found to harbour either a 72 kilobase pair (kB) plasmid coding for resistance to chloramphenicol, ampicillin, sulphonamide and tetracycline or a 65 kB plasmid coding for resistance to chloramphenicol, ampicillin and sulphonamide. Both plasmids yielded virtually indistinguishable restriction digest patterns. This suggests that the tetracycline resistance gene (Tc gene) is a non-essential component of one basic plasmid responsible for the multiple antibiotic resistances seen in the strains recovered during the outbreak. This illustrates the value of plasmid profiles to compare strains of non-encapsulated H. influenzae, and suggests that plasmid restriction enzyme analysis is critical.

Gentamicin resistance in clinical isolates of Escherichia coli encoded by genes of veterinary origin.

Seven (27%) of 26 gentamicin-resistant human clinical isolates of Escherichia coli were resistant to the veterinary aminoglycoside antibiotic apramycin. A gentamicin-resistant Klebsiella pneumoniae isolate from a patient infected with gentamicin/apramycin-resistant E. coli was also resistant to apramycin. DNA hybridisation studies showed that all gentamicin/apramycin-resistant isolates contained a gene encoding the enzyme 3-N-aminoglycoside acetyltransferase type IV (AAC[3]IV) that mediates resistance to gentamicin and apramycin in bacteria isolated from animals. Seven of the eight gentamicin/apramycin-resistant isolates were also resistant to the veterinary antihelminthic agent hygromycin B, a phenomenon observed previously in gentamicin/apramycin-resistant Enterobacteriaceae isolated from animals. Resistance to gentamicin/apramycin and hygromycin B was co-transferable in six of the isolates. Restriction enzyme analysis of plasmids in apramycin-resistant transconjugants derived from E. coli and K. pneumoniae isolates from the same patient were virtually identical, suggesting that inter-generic transfer of plasmids encoding apramycin resistance had occurred in vivo. These findings support the view that resistance to gentamicin and apramycin in clinical isolates of E. coli results from the spread of resistant organisms from animals to man, with subsequent inter-strain or inter-species spread, or both, of resistance genes on transferable plasmids.

A Case-Control Study of Nosocomial Ampicillin-Resistant Enterococcal Infection and Colonization at a University Hospital

To assess risk factors for colonization and nosocomial infection with ampicillin-resistant enterococci (ARE). Patients with ampicillin-resistant enterococci were compared retrospectively by logistic regression analysis with controls harboring susceptible strains. ARE were characterized by whole plasmid DNA analysis and restriction enzyme analysis of plasmid (REAP) DNA with EcoRI. The study was done at a 1,125 bed, tertiary-care teaching hospital in North Carolina with patients from whom enterococci were isolated from June 1, 1989, to March 30, 1991. The final study group comprised 44 cases with nosocomially-acquired colonization or infection with ARE and 100 controls with ampicillin-susceptible strains. Clinical and epidemiological risk factors for ARE were abstracted by chart review. After controlling for age and site of infection, patients with ARE were more likely to have been admitted previously to our hospital and to have received third-generation cephalosporins and clindamycin. However, only advanced age and clindamycin therapy were independently associated with presence of ARE. REAP with EcoRI showed 20 groups of enterococci on 19 different wards. These results suggest that ARE are endemic and multifocal in origin in our hospital and that advanced age and use of clindamycin are important selective risk factors for ARE colonization and infection.

Biochemical and molecular characterization ofSalmonella entericaserovarberta, and comparison of methods for typing

Strains of Salmonella enterica serovar berta (S. berta) from Denmark and seven other countries have been characterized with the aim of developing a rational typing strategy in connection with outbreak investigations. Biotyping divided the strains into H2S-positive (90%) and H2S-negative (10%) biovars. Six percent of the strains were resistant to one or more antimicrobial agents. Eighty-eight percent of the strains carried plasmids and 52 different plasmid profiles were recognized. Six of the common plasmid sizes in these profiles were shown by restriction enzyme analyses to contain more than one plasmid species. More than 90% of the strains had the same ribotype with the restriction enzymes Sma I and EcoR I and the same whole cell protein profile. Outer membrane protein profiles and isoenzyme profiles were identical in all S. berta analysed. Plasmid profiling in combination with restriction enzyme analysis of plasmids seemed to be the most rational typing strategy for S. berta. The results indicated that S. berta strains regardless of geographical source or host are possibly clonal in nature.

Methicillin-Resistant Staphylococcus aureus in Extended-Care Facilities: Experiences in a Veterans Affairs Nursing Home and a Review of the Literature

To delineate the spread of methicillin-resistant Staphylococcus aureus (MRSA) in a nursing home care unit (NHCU), determine its consequences, and discuss this experience in the context of reports from other nursing homes. Observational and descriptive; routine and special surveillance for MRSA, including a facility-wide prevalence survey; characterization of MRSA isolates by disk diffusion and agar dilution susceptibility studies and restriction enzyme analysis of plasmid (REAP) DNA. A 120-bed skilled nursing facility that is an integral part of the Veterans' Affairs Medical Center (VAMC), Portland, Oregon. The patients are predominantly elderly men with severe underlying diseases and functional impairments. An asymptomatic carrier brought MRSA into the NHCU in December 1987. During the next 15 months, 24 additional MRSA cases were detected. A prevalence survey conducted in March 1989 indicated that 39 (34%) of the 114 patients and 8 (7%) of the 117 employees were colonized or infected with MRSA. All strains were resistant to ciprofloxacin. REAP DNA indicated that 37 of 41 strains recovered in the March survey had identical patterns. Although 16 episodes of MRSA infection occurred in NHCU residents during 1988 through 1989, the outbreak had little effect on overall patterns of infectious morbidity and mortality in the facility. The outbreak, however, did result in an increased MRSA caseload at the medical center's acute-care division. During the last three years, MRSA colonization and infection have become common in the NHCU at the Portland VAMC; this experience parallels that reported by several nursing homes in other parts of the country.

Methicillin-Resistant Staphylococcus aureus in Extended-Care Facilities: Experiences in a Veterans Affairs Nursing Home and a Review of the Literature

AbstractObjectives:To delineate the spread of methicillin-resistant Staphylococcus aureus (MRSA) in a nursing home care unit (NHCU), determine its consequences, and discuss this experience in the context of reports fi-om other nursing homes.Design:Observational and descriptive; routine and special surveillance for MRSA, including a facility-wide prevalence survey; characterization of MRSA isolates by disk diffusion and agar diluation susceptibility studies and restriction enzyme analysis of plasmid (REAP) DNA.Setting and Patients:A 120-bed skilled nursing facility that is an integral part of the Veterans' Affairs Medical Center (VAMC), Portland, Oregon. The patients are predominantly elderly men with severe underlying diseases and functional impairments.Results:An asymptomatic carrier brought MRSA into the NHCU in December 1987. During the next 15 months, 24 additional MRSA cases were detected. A prevalence survey conducted in March 1989 indicated that 39 (34%) of the 114 patients and 8 (7%) of the 117 employees were colonized or infected with MRSA. All strains were resistant to ciprofloxacin. REAP DNA indicated that 37 of 41 strains recovered in the March survey had identical patterns. Although 16 episodes of MRSA infection occurred in NHCU residents during 1988 through 1989, the outbreak had little effect on overall patterns of infectious morbidity and mortality in the facility. The outbreak, however, did result in an increased MRSA caseload at the medical center's acute-care division.Conclusions:During the last three years, MRSA colonization and infection have become common in the NHCU at the Portland VAMC; this experience parallels that reported by several nursing homes in other parts of the country.

Restriction enzyme analysis of plasmid DNA and bacteriophage typing of paired Staphylococcus aureus blood culture isolates

We compared restriction enzyme analysis of plasmid (REAP) DNA profiling with bacteriophage typing for determination of similarities and differences among 50 pairs of Staphylococcus aureus blood isolates from patients with multiple positive blood cultures. Isolates from 17 pairs did not have detectable plasmids. Isolates from 33 pairs had plasmids classified into 17 distinct REAP DNA profiles. Paired isolates from 31 of these episodes were identical to one another. By phage typing, 35 pairs had strong lytic reactions to a phage(s), 9 pairs lacked strong reactions, and 6 pairs consisted of a strongly reactive isolate and an isolate with no strong reaction to a phage. When consolidated into 11 general phage groups, pairs from 44 of the 50 episodes were in the same general group. REAP DNA profiles were highly reproducible (99%), whereas phage typing was not. REAP DNA profiling is superior to phage typing as a technique for determining similarities and differences among S. aureus blood isolates.