الفهرس 
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Abstract
With the increased use of antibiotics to treat bacterial infections,
pathogenic strains have acquired antibiotic resistance, causing a major
problem in modern therapeutics. Among the medical problems being
greatly affected by the problem of the widespread of antimicrobial
resistance is the urinary tract infection (UTI). UTI is a very common
infection both in the community and among hospital patients.
Escherichia coli is the most common cause of uncomplicated UTI
followed by Staphylococcus saprophyticus then other aerobic gram
negative rods, such as Klebsiella, Pseudomonas and Proteus species.
Aminoglycoside antibiotics (AGAs) are aminocyclitols that kill bacteria
by inhibiting protein synthesis. They are among the antibacterials that are
highly recommended for treatment of UTIs. As with other drugs, their
overuse and misuse lead to development of resistance in many important
pathogens. Three mechanisms are known to be responsible for bacterial
resistance to aminoglycoside antibiotics: first, decreased intracellular
accumulation by outer membrane permeability alteration, diminished
inner membrane transport, or active efflux; second, target modification
by mutation of 16S rRNA or ribosomal proteins coding genes or by 16S
rRNA methylation, a mechanism newly identified in clinical isolates; and
third, enzyme-mediated drug modification resulting in compromised
binding to target site, the most prevalent in clinical setting.
The current study aimed at determination of resistance percentages
and patterns of uropathogens of the genera Escherichia, Klebsiella, and
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Pseudomonas, isolated from hospitalized UTI patients, to aminoglycoside
antibiotics and to detect the most prevalent plasmid-mediated
aminoglycoside modifying enzymes (AMEs) and 16S rRNA
methyltransferases.
Atotal of 150 uropathogenic isolates were recovered from urine
specimens of hospitalized UTI patients and identified by microbiological
methods. Of these isolates, nine isolates (6.0%) were found to be Gram
positive cocci and 141 isolates (94.0%) were found to be Gram negative
rods. Of these Gram negative rods, 73 E. coli isolates and 24 Klebsiella
spp. isolates were identified by testing for indole production, citrate
utilization and reactions on TSI agar. Thirteen isolates showed growth
with green pigment production on cetrimide agar and positive oxidase
test and consequently identified as Pseudomonas species.
All the recovered uropathogens were tested for their susceptibility to
gentamicin, tobramycin, amikacin, neomycin, netilmicin, and kanamycin
by disc diffusion method. An overall resistance percentage of 57.2% was
detected to at least one of the tested AGAs. Of which, 17.4% were
resistant to one AGA, 31.7% were resistant to two AGAs, and 50.7%
were resistant to three or more AGAs. Of all tested AGAs, the highest
resistance percentage was detected to kanamycin (53.6%) and lowest
resistance percentage was to amikacin (7.2%). Resistance percentages of
33.6%, 23.6%, 24.5%, and 14.5% were detected to gentamicin,
tobramycin, neomycin and netilmicin, respectively. Antibiogram analysis
of E. coli isolates showed 56.1% resistance to at least one of the tested
AGAs. The highest resistance was to kanamycin followed by neomycin,
gentamicin, tobramycin, netilmicin then amikacin with resistance
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percentages of 52.0%, 31.5%, 28.7%, 16.4%, 6.8% then 4.1%,
respectively. In Klebsiella spp. isolates, 41.6% were resistant to at least
one of the tested AGAs. About 37.5% of all Klebsiella spp. isolates were
resistant to kanamycin, 33.3% were resistant to gentamicin, 20.8% were
resistant to tobramycin, 12.5% were resistant to netilmicin, 4.1% were
resistant to neomycin and 4.1% were resistant to amikacin. A
significantly high resistance to AGAs was detected in Pseudomonas spp.
isolates. About 92.3% of them were resistant to at least one of the tested
AGAs. Pseudomonas spp. isolates had shown highest resistance (92.3%)
to kanamycin and lowest resistance (23.0%) to neomycin, 69.2%, 61.5%,
61.5% and 30.7% resistance to tobramycin, gentamicin, netilmicin and
amikacin, respectively.
A total of 17 resistance phenotypes were identified from the
antibiogram analysis of the tested isolates. E. coli isolates exhibited
thirteen resistance phenotypes. Seven resistance phenotypes were
identified in Klebsiella spp. isolates while six were identified in
Pseudomonas spp. isolates.
Plasmids were extracted from isolates having reduced susceptibility
to at least one of the tested aminoglycoside antibiotics. Detection of two
plasmid-mediated resistance mechanisms, enzyme modification and 16S
rRNA methylation, was carried out by PCR assay using plasmids as
DNA templates.
Five sets of primers were designed for PCR detection of the AMEscoding
genes: aph(3’)-I, aac(6’)-I, aac(3)-I, aac(3)-II and ant(2’’)-I in all
resistant isolates. AMEs-coding genes were detected on the plasmids of
93.6% of all resistant isolates. Individual AMEs-coding genes were
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detected in 44.4% of isolates, while combinations of two and three
AMEs-coding genes were detected in 20.6% and 28.5% of isolates,
respectively. A total of nine different AMEs-coding genes combinations
were detected in all resistant isolates.
Of all tested AMEs-coding genes, ant(2’’)-I was the most frequently
encountered gene (53.9%) followed by aac(6’)-I and aac(3)-II each were
found in 24 isolates (38%). Twenty-one isolates (33.3%) were found to
carry aph(3’)-I, while aac(3)-I gene was not detected in any of the tested
resistant isolates. Regarding the distribution of the four AMEs-coding
genes in resistant isolates of different genera, ant(2’’)-I was detected in
46.3% of E. coli resistant isolates, aph(3’)-I in 44.1%, while 34.1% and
31.5% of them carried aac(3)-II and aac(6’)-I, respectively. In resistant
Klebsiella spp. isolates, ant(2’’)-I was the most commonly detected
(70.0%), followed by aac(3)-II (60.0%), aac(6’)-I (40.0%), while
aph(3’)-I was the least commonly detected in 20.0% of Klebsiella spp.
resistant isolates. The prevalence percentages of the four AMEs-coding
genes in resistant Pseudomonas spp. isolates were 66.6%, 58.3, 33.3%
and 16.6% for ant(2’’)-I, aac(6’)-I, aac(3)-II and aph(3’)-I, respectively.
None of the aforementioned genes was detected on the plasmids of only
four resistant isolates (6.3%) despite showing resistance to at least one of
the tested AGAs.
Four PCR products representing the four genes (aph(3’)-I, ant(2’’)-I,
aac(6’)-I and aac(3)-II) were sequenced from both directions by
Macrogen company, Korea. Analysis of the translation product of the
final sequences showed 100% similarities with their homologous
enzymes from different bacterial species.
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16S rRNA methylation was the second resistance mechanism
screened in only the six isolates that showed resistance to all tested
members of the 4,6 disubstituted 2DOS-containing AGAs. 16S rRNA
methylases were detected phenotypically by testing the susceptibility of
the multiresistant isolates to both streptomycin and apramycin. None of
the six multiresistant isolates exhibited the N1-A1408 16S-RMTases
resistance phenotype and only one of them exhibited that of N7-G1405
16S-RMTases. Two isolates were resistant to all tested AGAs. As the
resistance phenotype may be complicated by co-existance of other
resistance mechanisms, all isolates that showed resistance to all tested 4,6
disubstituted 2DOS-containing AGAs were screened for 16S RMTasescoding
genes by PCR assay.
Screening the plasmids of the six multiresistant isolates for the two
16S RMTases-coding genes armA and rmtA showed that, none of them
carried any of the tested genes.
Finally, plasmid localization of resistance genes and their ability to
transfer was tested by transformation assay using E. coli DH5α as a
recipient strain. Transformation experiments were successful in 11.1% of
resistant ones. The transformation was confirmed by antimicrobial
susceptibility testing and by PCR assays. The resistance profiles were
identical in six transformants and donor clinical isolates. In only one
transformant (116T), resistance to gentamicin was lost as compared to the
donor clinical isolate resistance profile. ant(2’’)-I gene was not detected
in this transformant (116T) despite being detected in the donor isolate
(116).
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This study demonstrated a high dissemination of plasmid-mediated
aminoglycoside resistance in relevant uropathogens. The threat of
horizontal gene transfer necessitates the implementation of proper
infection control measures in hospitals. It is also recommended that new
guidelines have to be undertaken in Egypt to limit or prevent the misuse
and abuse of antimicrobial agents.