dc.description.abstract | Tetracyclines are still the second most commonly used antibiotics
worldwide, after penicillins. However, their efficacy against a variety
of bacterial and rickettsial disease agents has been reduced by the
high frequency of microbial resistance. Initially, organisms isolated
from patients seemed to be resistant to one antibiotic. From the
1950's multiple-drug-resistant organisms were noted.
The objectives of the present study were:
(1) To determine the minimum inhibitory concentrations of
oxytetracycline hydrochloride to S. typhimurium strains isolated
from man and cattle in Kenya.
(2) To identify tetracycline resistance genes belonging to the types A,
B or C amoDg the strains.
(3) To determine the location of these genes in the S. typhimurium
strains.
(4) To determine the total plasmid content of the strains for use as
epidemiological tools.
Salmonella typhimurium strains from man and cattle were
examined for tetracycline resistance genes using synthetic probes A,
B, and C Ninety-seven (97) strains isolated from either blood, stool,
urine, pus or cerebrospinal fluid of patients at Kenyatta National
Hospital during the period 1988-90, and 17 strains isolated from cases
of calf diarrhea reported to the Department of Veterinary Pathology
and Microbiology, University of Nairobi, were used in this study.
The determination of minimum inhibitory concentration (M.LC)
was done using the agar dilution method. Strains with M.LC
>5Ilg/ml were categorized as resistant to oxytetracycline.
Ninety-three percent (93%) of the human strains and 83% of the
bovine strains were resistant. In general, resistant bovine strains had
higher M.LC.s than human strains. Ten percent (10%) of the strains
were sensitive to oxytetracycline.
Plasmid isolation was carried out by the principle based upon the
disruption of the bacterial cell wall by treatment with lysozyme, lysis
of the internal cell membranes with detergent, and denaturation of
chromosomal DNA by alkaline pH. Plasmid DNA was recovered by
ethanol precipitation in the cold. Plasmids of various molecular
weights were separated by electrophoresis. Total plasmids were
classified into 6 plasmid profile groups on the basis of molecular
weights. Fourteen (14) strains lost plasmids during subculturing and
subsequent plasmid isolation procedures. The major (heavy)
plasmids were 65, 63(60), 46, and 36 megadaltons (Mda). Plasmid
profile analysis indicated that group 2 <major plasmids 65 and 46
megadaltons) contained 37(40%) of the human strains and 4(25%) of
the bovine strains.
Colony and Southern blot hybridization were carried out using 'Y
32P-labelled synthetic tetracycline resistance probes A, B, and C on
synthetic nylon filters.
Colony blot hybridization results indicated 22 strains (19 human
and 3 bovine) were positive for tetracycline resistance determinant
type A, 7 and 5 human strains tested positive for presence of
tetracycline resistance determinants type Band C respectively. Sixty
percent (60%) of the resistant strains were negative for resistance
determinants A, B or C and may belong to other tetracycline
resistance determinants. Three (3) human strains had determinants
encoding both type A and B tetracycline resistance.
Southern blot hybridization results revealed a 65 (also the 63 and
60) Mda plasmids and a smaller 5.2 Mda plasmid as responsible for
carrying the resistance determinants A, Band C encoding
tetracycline resistance. An apparently cryptic plasmid 4.0 MDa was
found in all the human and bovine strains that had plasmids. This
plasmid may be an important epidemiological marker.
There was no consistent connection between plasmid profile
groups and M.I.e. figures for the strains. However, plasmid profile
group 2 (major plasmids 65 and 46 Mda) contained 60% of all strains
that had the resistance determinant type A. This same group showed
25% similarity in total plasmid content of human and bovine strains
and may serve to emphasize the possibility of clonal origin of
human and bovine S. typhimurium strains.
All the strains that tested positive for presence of tetracycline
resistance determinant type Band Chad M.I.e. figures >64Jlg/ml.
However, there was no consistent relationship between M.Le.
figures and resistance determinant type A.
The findings of this study indicate that plasmid profiling is a
useful tool for characterizing strains from common sources and the
spread of such strains and their antimicrobial resistance
determinants. A remarkably high level of resistance by human and
bovine strains to oxytetracycline was observed. There exisfs a
potential cross-transfer of resistance among human and bovine
populations as evidenced by the results of the Southern blot
hybridization. The emergence and transmission of resistance factors,
even among the once tetracycline-sensitive microbial strains, may
result in treatment failure and subsequent rise in treatment costs. | en |