Title page for ETD etd-511132839711171

Type of Document Dissertation
Author Jensen-Cain, Donna Marie
Author's Email Address shamrock@vt.edu
URN etd-511132839711171
Title Macrolide Resistance in Mycobacterium avium
Degree PhD
Department Biology
Advisory Committee
Advisor Name Title
Dean, Dennis R.
Rutherford, Charles L.
Wilkins, Tracy D.
Winkel, Brenda S. J.
Falkinham, Joseph O. III Committee Chair
  • Mycobacterium avium
  • clarithromycin
  • azithromycin
  • antibiotic-resistance
Date of Defense 1997-04-16
Availability unrestricted

Mycobacterium avium isolates resistant to clarithromycin

and azithromycin have been recovered from patients

undergoing antibiotic therapy. Comparison of DNA

fingerprints of sensitive and resistant isolates showed that

resistance resulted from mutation of the original, sensitive

isolate in five of seven patients. In the other two patients,

the clarithromycin-resistant isolates were unrelated to the

sensitive isolate, suggesting that the resistant isolate resulted

from either superinfection or selection of a resistant strain

from a polyclonal population.

Investigation of the mechanisms of clarithromycin and

azithromycin resistance in M. avium showed that high-level

resistance resulted from a point mutation at position

A-2058 in the 23S rRNA. Based on this finding, a rapid

screen for clarithromycin-resistance in M. avium was

developed based on PCR. Twenty-three clinical isolates

were analyzed, seven of which were

clarithromycin-resistant. The target product was amplified

only in clarithromycin-resistant strains, all of which had

mutations at position 2058.

A polyuridylic acid (poly U)-dependent in vitro translation

system from M. avium was developed to investigate the

effect of antibiotics on protein synthesis. Clarithromycin

was an effective inhibitor of protein synthesis in cell-free

extracts of a susceptible M. avium strain, whereas a

high-level resistant strain was less susceptible to

clarithromycin in vitro. Mixtures of extracts from sensitive

and resistant strains showed a pattern of clarithromycin

inhibition similar to the resistant strain, suggesting that

resistance may be dominant in partial diploids.

Three M. avium strains exhibiting step-wise, intermediate

resistance to azithromycin were characterized in

comparison to the sensitive parent. All strains were similar

in hydrophobicity, growth medium requirements, and

growth response to temperature. The

azithromycin-resistant strains were resistant to several

unrelated agents, including ciprofloxacin, rifabutin, and

ethidium bromide. Addition of carbonyl cyanide

m-chlorophenylhydrazone (CCCP) did not lower minimal

inhibitory concentrations (MICs) for ciprofloxacin or

ethidium bromide. Cell-free extracts of the strains were as

sensitive to azithromycin in vitro as the parent strain. The

results rule out inactivation, efflux, and mutations in the

target as resistance mechanisms, and suggest intermediate

resistance may be due to altered permeability of the cell

wall or membrane.

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