Genetic and physical characterization of IncM plasmid pBWH1 and its variance among natural isolates

Abstract

We present a genetic and physical characterization of the IncM plasmid pBWH1. A physical map was constructed for the enzymes EcoRI, BamHI, Sall, BglII, HindIII, MstII, and XhoI. A series of deletions and a series of subclones of pBWH1 were constructed and used to determine the locations on this map of the transfer region; the replication region; and the genes determining resistance to beta-lactamg, chlotaamphenicol, the sulfonamides, and gentamicin. We compared 51 different isolates, including isolates which had lost individual antibiotic resistances or the transfer phenotype, and showed that variations occureed in all regions of the plasmid genome. Frequently, correlations could be made between phenotypic variation and variation of the EcoRI fragments which contained the gene determining that phenotype.The plasmid pBWH1 is a 92-kilobase IncM plasmid that has a broad host range for gram-negative members of the family Enterobacteriacae and carries four antibiotic resistance genes. These genes encode resistance to the aminoglycosides gentamicin, tobramicin, and kanamycin; to ampicillin and carbenicillin; to chloramphenicol; and to the sulfonamides (16). We have been monitoring the evolution of this plasmid and its derivatives at one location. Over a 10-year period, we obtained over 2,000 different isolates of strains carrying this plasmid as we followed the epidemic spread of pBWH1 at the Brigham and Women's Hospital in Boston.We examined phenotypic changes and restriction fragment variations. Analysis of these variations in pBWH1 may extend an important technique in the field of molecular evolution. Restriction fragment variation of, for example, metazoan mitochondria is currently being used to ascertain phylogenetic relationships (7, 13). The results of these experiments can be compared with the relationships inferred from comparative morphology and the fossil record; but rholecular data are available only for the present. The molecular evolution of bacterial plasmids is much faster, with significant changes appearing over relatively brief periods. Thus it is possible to monitor changing populations of plasmids through real time, as well as to extensively sample a population at any given time.pBWH1 has been found in strains of six different genera of the family Enterobacteriacae. Screening of many of these isolates has shown that the full phenotype of pBWH1, as well as a standard pattern of EcoRI restriction enzyme fragments, has persisted from 1976 to the present. A number of isolates, however, have shoWn significant phenotypic variations, losing resistance to an antibiotic or the ability to transfer the plasmid. There have also been many variations in the pattern of EcoRI fragments. We refer to these isolates as the pBWH1 series of plasmids. In this report we present a genetic and restriction enzyme map of pBWH1 and discuss variations in natural isolates in relationship to this map. We * Corresponding author.show that the variations of EcoRI restriction enzyme sites indicate that variations occur throughout th...

Nosocomial spread of an amikacin resistance gene on both a mobilized, nonconjugative plasmid and a conjugative plasmid

“…Amikacin resistance outbreaks that have been reported in the past often involved single plasmid species (12,43,45). The outbreak we report here involves four plasmid species, with two predominating.…”

“…These genes began to appear in hospital isolates only after gentamicin had been widely used for some years, with one gene often spreading through one or a number of hospitals on a single plasmid (8,12,28). One gene has tended to predominate in one hospital or in the hospitals of a region, but gentamicin resistance remains rare in strains of the Enterobacteriaceae in many hospitals of some regions and was observed in the community flora in only one of three regions recently surveyed (18,27).…”

Molecular evolution, species distribution, and clinical consequences of an endemic aminoglycoside resistance plasmid

“…This (21). The correlation was confirmed in all of 52 random isolates selected by these criteria at this center over the 6-year period by always finding in transconjugants, transformants, or direct extracts of each the restriction fragments characteristic of pBWH1 (8,16).…”

In vitro studies on long‐term stability of R plasmids in Escherichia coli K12

“…Molecular changes in plasmids were also regularly found in cases where the original plasmid could be traced over several years in different hosts (LEE et al 1984, HOPKINS et al 1986, M~Y E R et al 1986). The considerable long-term stability of distinct plasmids must be emphasized.…”