VOL. XXX NO. 8
THE JOURNAL OF ANTIBIOTICS
RESISTANCE ANTIBIOTICS:
OF BACTERIA
TO THE NEWER
AN EPIDEMIOLOGICAL
AND
655
AMINOGLYCOSIDE ENZYMATIC
STUDY
MARLYSE DEVAUD,FRITZ H. KAYSERand URSUL.A HUBER Institute of Medical Microbiology, University of Zurich CH-8028 Zurich, Switzerland (Received for publication May 8, 1977) Four hundred and thirty-five Staphylococcus aureus strains and 395 strains of gramnegative bacteria were tested for susceptibility to gentamicin, sisomicin, tobramycin, netilmicin and amikacin. None of the staphylococcal strains were resistant to the 5 drugs, except two strains that were resistant to amikacin. Eighteen percent of the gram-negative bacteria were resistant to gentamicin, 16% to tobramycin, 14% to sisomicin, 7 % to netilmicin and 2 % to amikacin. Tests from representative strains showed that these differenceswere mainly due to the production of aminoglycoside-modifyingenzymes. The lower incidence of strains resistant to sisomicin compared to gentamicin was due to a slightly better antimicrobial activity of sisomicin. Results of experiments on the efficiencyof enzymatic phosphorylation, acetylation and adenylylation, and the inactivation of aminoglycoside antibiotics were not always in concurrence with the degree of phenotypic expression of resistance. Thus, the aminoglycoside 3'-phosphotransferase of staphylococci modified amikacin, but the strains were susceptible to the drug. Similarly, the aminoglycoside 2"-nucleotidyltransferase from members of Enterobacteriaceae and Pseudomonas efficiently adenylylated and inactivated netilmicin, but the strains were susceptible to netilmicin. On the other hand, there can be factors, intrinsically inherent in some strains, contributing to their phenotypic expression of resistance level. It is inferred, therefore, that the contribution of enzymes to the degree of resistance to aminoglycoside antibiotics in a given strain can be evaluated only by comparative examination of resistance in the wild type strain and its corresponding enzyme-negativevariant. In recent years gentamicin, with or without the concomitant administration of beta-lactam antibiotics, has been used with increasing frequency in the treatment of hospital-acquired,
gram-negative
bacterial infections. This resulted in increasing emergence of gentamicin-resistant strains.
The situa-
tion is clearly demonstrated in Table 1, which contains the total number of strains examined at our institution from 1974 to 1976 and the percentage of gentamicin-resistant
isolates. Since the Institute
of Medical Microbiology in Zurich has to perform diagnostic microbiology for the University Hospital and for about 30 smaller hospitals in the area and has to serve also as a Public Health Laboratory for the Kanton of Zurich, these data can be considered to represent the prevailing situation in the eastern part of Switzerland. The frequency of 9 % of gentamicin-resistant hospitalized patients in 1976 is appallingly high.
cultures isolated from
Because resistance to gentamicin occurred mostly in strains having resistance to many other antibiotics a comparative examination of the newly developed or recently introduced aminoglycoside antibiotics seemed appropriate and necessary. This paper presents the result of examination of susceptibility of 830 gram-positive and gramnegative bacterial strains of clinical origin to five selected aminoglycoside antibiotics.
In addition,
data on susceptibility to modification of these antibiotics by enzymes isolated from representative gentamicin-resistant
strains, are presented.
These data help in explaining differences in resistance
656
THE
Table
1. Number of strains examined of gram-positive and gram-negative
JOURNAL
and percentage bacteria.
Relation to hospitalizationa
Bacteria
OF
ANTIBIOTICS
of gentamicin
AUG.
resistant
1974 (May-December) No. of strains
cultures
of clinical
1975
Genrb %
1977
isolates
1976
No. of strains
GenRb %
No. of strains
Genjtb %
stllph _vlococcus aureus
Outpatients
170
0
406
0
459
Hospitalized
pat.
1,355
0
2,685
0
2,639
Staple lococcus epidernlidis
Outpatients Hospitalized
168
1
247
0
183
pat.
1,239
1
1,899
0
1,114
02
Escherichia coli
Outpatients Hospitalized
450 2,215
0
1,130
0
0
4,742
1
1,199 4,724
0
pat.
11
0
12
0
13
0
54
0
96
5
83
6
92
0
200
1
191
4
pat.
761
6
1,886
12
1,875
18
37
0
72
0
pat.
230
5
524
8
2
0
17
13
31
pat.
31
35
145
59
529
80
0
219
0
1
1,166
1 1
239
pat.
101 548
1,154
3
7
51
61
0
5
291
383
6
Outpatients
Ci trobacte r freundii
Hospitalized
pat.
Outpatients
Klebsiella spp.
Hospitalized
Enterobucter spp.
Outpatients Hospitalized Outpatients
Serratia spp.
Hospitalized
6
0
1
2
98 571
I 9
Protcus nlirabilis
Outpatients Hospitalized
Proteus spp. indole-positive
Outpatients Hospitalized
pat.
14 150
Proteus inconstalls
Outpatients Hospitalized
2
50
3
33
pat.
15
53
24
62
27
63
57 79
0
48
0
46
0
pat.
0
68
1
71
7
Pseudonroncrs spp.
Outpatients Hospitalized
3
223
9
2,143
3 9
212
pat.
86 1,069
Further non-fermentative gram-negative rods
Outpatients Hospitalized
61 176
7
87
pat.
10
302
Total
Outpatients Hospitalized
1,249 7,921
1
2,703
91
2,813
pat.
4
15,971
6
15,712
Outpatients
Salmonella spp.
Hospitalized
a:
Status
b:
GenR=gentamicin
to gentamicin,
Bacterial
of prior hospitalization
sisomicin,
of outpatients
2 5
2
2,231
9 14
96
15
311
15
9
not determined.
resistance.
tobramycin,
netilmicin
and amikacin
Materials
and Methods
existing
in our collection
of strains.
Strains
These were isolated in 1976 from different clinical materials. They were comprised of Staphylococcus aurcus (435); Eschericlria coli (114); Klebsiella pneumoniae (71); Klebsiella ozaenae (4); Serratia marecscens (43); Elrterobaeicr cloacae (36); Enterobacter agglomerans (1); Citrobacter freundii (6); Proteus vulgaris (10); Proteus mirabilis (9); Proteus rettgeri (3); Proteus morganii (3); Proteus inconstatts (3); Salmonella spp. (15); Shigella spp. (2); Pseudomonas aeruginosa (57); Pseudomonas mcrltophilia (3); Pseudomonas spp. (2); Acinetobacter spp. (5); Alcaligenes spp. (5). All strains were identified according to standard procedures. Staph. aureus 5532 was a gentarnicin-resistant strain, isolated by PORTHOUSE et al.1) in England.
VOL. XXX NO. 8
THE JOURNAL OF ANTIBIOTICS
657
Staph. aureus S25 was a gentamicin-susceptible mutant of 5532. Both strains were provided by Dr. BROWN. E. coli W677 (pHJR66) was a gift of Dr. SMITH.2)This strain is the source of the adenylylating enzyme, widely used for enzymatic assay of gentamicin. All the rest of the strains were of wild type, isolated at our institution. Antibiotics and Chemicals. Laboratory standard preparation of gentarnicin C complex, sisomicin and netilmicin were generously supplied by Schering Corp. (Bloomfield, N.Y.); tobramycin by E. Lilly and Co. (Indianapolis, Ind.), and arnikacin by Bristol Lab. (Syracuse, N. Y.). Radio-labelled chemicals were bought from the Radiochemical Centre (Amersham, England). Dithiothreitol, ATP and acetyl-CoA were obtained from Calbiochem (Los Angeles, Calif.), and lysostaphin from Becton, Dickinson and Co. (Orangeburg, N. Y.). Inorganic salts of analytical grade were bought from Merck (Darmstadt, Germany), and agarose from Behringwerke AG (Marburg, Germany). Antibiotic Susceptibility Testing. Disc susceptibility testing was performed by the slightly modified single-disc method of BAUER et a1.3) and the U.S. Food and Drug Administration,4,5) using MUELLER-HINTON agar of Baltimore Biological Laboratories (BBL) (Cockeysville, Md.) supplemented with 5%, (v/v) old human blood from the blood bank. An inhibition zone of 15 mm or more around the 10ug gentamicin disc, for a given strain, was interpreted as being susceptible to the drug." The minimal inhibitory concentration (MIC) of drugs was determined on MUELLER-HINTON agar (BBL) according to the standard methods proposed by ERICSSON and SHERRIS." Production of Enzyme. The shock method of NossAL and HEPPEL8)was used for gram-negative organisms. The bacterial strains under study were serially subcultured on Brain Heart Infusion (BHI) agar (Difco), containing 10 pg gentamicin/ml, and inoculated into a medium composed of Bacto-tryptone (Difco), 10g; Bacto yeast extract (Difco), 50 g; NaCI, 10 g; glucose, 4 g; and an amount of water sufficient to make I liter. The pH was adjusted to 7.2. A hundred ml of the medium was inoculated with one ml of a 24-hoursold culture and incubated in a shaking water bath at 37'C until the end of logarithmic growth phase. Cells were harvested and washed twice with 40 ml of a buffer containing 0.01 M Tris-HCl and 0.03 M NaCl, pH 7.8 and were suspended in 20 ml of 0.033 M Tris-HCl buffer, pH 7.3 containing 0.003 M EDTA and 20% (w/v) sucrose. After it stood at room temperature for 10 minutes, the suspension was stirred with a magnetic stirrer for 5 minutes and centrifuged for 15 minutes at 10,000 x g and 4°C. The supernatant was discarded and the remaining sucrose was removed carefully from the wall of tubes with cotton swabs. The pellet of cells was suspended in 4 ml of an ice-cold solution of 0.0005 M MgCl2, stirred for 5 minutes and centrifuged for 15 minutes at 22,000 x g and 4oC. The supernatant (osmotic shockate) was carefully decanted to a flask and stabilized with 0.001 Mdithiothreitol. Enzymatic extracts of staphylococci were obtained either by the shock method or by lysing the cells with lysostaphin as previously described.9) Enzymatic Assays. ssays. These were similar to those described by BENVENISTE et al.10), DAVIESet al.11) and HAAS and DAVIES.12)Inactivation by adenylylation of aminoglycosides was tested in a reaction mixture consisting of 40 /Il enzymatic extract; 3 /smol of Tris-maleate buffer, pH 7.4; 1.4 /cmol MgCl2; 0.1 ,umol dithiothreitol; 40 nmol (2-3H)ATP (specific activity, 60uCi/,umol) and I nmol of drug base in a total volume of 130 lil. Inactivation by phosphorylation was tested in a reaction mixture containing 40 nmol of (y-52P)ATP (specific activity, 1 - 4 mCi/mmol). Acetylation was examined in tests comprising 5.5 nmol (1-11C)acetyl-CoA (specific activity, 36.3 mCi/mmol) and 0.4 nmol drug base. Appropriate controls were devoid of either the labelled coenzymes, or the drug base or the enzymatic extract. After incubating for 2.5 hours at 35°C, 501d were assayed for residual antibiotic by agar diffusion13) in Antibiotic Medium 5 (Difco) containing agarose instead of agar. Bacillus subtilis ATCC 6633 was used as test organism, in some cases, and the enzyme-producing strain was used in certain others. Another 50 pl were examined for the extent of adenylylation, phosphorylation or acetylation by mea-
658
THE
suring
the
and
radioactivity
incorporated
coworkers.11)
Instr.,
were
Grove,
appropriately 50ul
were
conditions
was
Ill.)
determining
which
by
Radioactivity
Downers For
JOURNAL
in
the
relative
diluted
and
assayed
ensured
10
ml
of
the the
linear
in
of
5
paper
always
described
by
spectrometer
1977
DAVIES
(Packard
scintillant. as
incubated
phosphocellulose
assay
scintillation
aminoglycosides
mixtures
were
binding
Tri-Carb
based the
AUG.
paper
a Packard
a toluene
reaction
assays
ANTIBIOTICS
phosphocellulose
counted
efficiency
by
that
the
OF
for
substrates,
2.5
binding
and
enzymatic
method
5 minutes
extracts
at
mentioned
35'C,
after
above.
These
occurring.
Elimination. Attempts
to
previously14).
obtain To
method
was
enzyme-negative
eliminate
variants
R-plasmids
of
from
Staphylococcus
strains
gram-negative
bacteria,
were the
made
sodium
as
described
dodecyl
sulfate
used.15)
Results
Antimicrobial The from
in
our
vitro
activity
of
collection
concentration
of
(MIC)
No
strain to
area
of
The
susceptibility
collection
1).
against
Shigella,
are
to
pattern
these
time,
Tables
shown.
of
strains
435
The
(S.
MITSUHASHI, The
and
data
to
contrast
ed
and
Some
strains
1976),
possessed
an
2 to
amikacin
(see
mutant
and
affinity
inhibitory
reported
by
outlined
in
was
however,
other
Table
observed do
3. to
occur
be
in
our
the
ring
III
that
of
In
tobramycin This Table
and is
the 5).
were
position
of
resistant
resistance in
still
and
addition
to
reason
for
The the
rather
I
time
Zurich
5).
(see
of
Fig. and
strain
1)
Chemotherapy
strains.22,23)
drug,
however,
APH(2")
intermediate produced
was
still
produced
MIC
of
tobramycin
is is
by
to
the and
sub-
FK170)
observed hydroxyl
acetylating
1).
amikacin.
APH(2")
resistance an
weak
(i.e.
recently the
producing
a to
amikacin was
Fig.
phosphorylat-
susceptible to
This
(see
also,
phosphorylates Strains
5532
Nomencla-
of
aminoglycosides
resistant The
are
Japan).
The
enzyme
Plasmid
epidermidis
amikacin,
moderately
The
high
the
observed.
antibiotics
the
phenotypically
unknown.
enzyme
in
Society
Staph.
ring
Table
tobramycin,
amikacin.26)
of
were
(see
APH(2"),
from
organisms,
England.1,24)
aminoglycosides.25)
taken
and 3
or
At was
aminoglycoside
Maebashi,
aureus
(see
4.
appeared
International
therefore,
Table
gentamicin
strains
modifying were
gram-negative
enzyme
APH(3')-4,
sisomicin 5).
in
staphylococcal
mechanism
Table
(see
group
in to
Enzymes of
University, Staph.
shown
aureus
abbreviations
in
of
staphylococci
of
mutant. strain
those
collection
resistant
Chemoresistance, Gunma
found
hydroxyl
gentamicin,
to
selected
minimal
as
species,
is
isolate
capable
the on
recently
staphylococci
resistance
negative
was
producing
inherent
position
low
4
by
gentamicin-resistant in
to
the
these
strains
no
Modifying
strains
commission
the
strains of
with
3'-phosphotransferases
inactivated and
The
species in
in
organisms,
2.
similar
Citrobacter
aureus
March
communication,
type
the
tests
the
personal
to
strate,
are
Staphylococcus
nomenclature
phosphorylates
In
on
from
APH(3')
enzyme
Table
bacterial
gentamicin
gentamicin-resistant
enzymatic
Group21)
strains
Staphylococcus
(January
however,
5•`7,
ture
gram-negative
in
among and
Resistance
395
summarized
found
Aminoglycoside In
against
susceptible
were Enterobacter
drugs.
Aminoglycosides
1).
of
that
isolates,
drugs
of
antibiotics
strains
examined
Table
Table
the
Salmonella, the
(see
Since
clinical
Resistant
of
resistant
fresh
of
investigators16•`20).
Activity
5 aminoglycosidic
in group
exhibited netilmicin enzyme
and with
phosphotransferaseamikacin
against
the
VOL.
XXX
Table
NO.
8
THE
JOURNAL
2. Summary of antimicrobial bacteria (Zurich, 1976).
activity
Susceptible Antibiotic
No. of strains
OF
659
of five aminoglycoside
antibiotics
strainsa
against
Resistant
Geometric mean of M ICS (,ug/ml)
% of strains
ANTIBIOTICS
Range
of
MICs
No. of strains
% of strains
70 54
17.7 13.7 15.9 7.3 2.0
325 341
82.3 86.3
0.93 0.56
0.2•`6.25
Tobramycin Netilmicin
332 366
84.1 92.7
0.80 0.84
0.1•`6.25 0.1•`6.25
63 29
Amikacin
387
98.0
1.66
0.4•`12.5
8
0.1•`6.25
strains
Geometric mean of MICs (,ug/ml)
(hg/ml)
Gentamicin Sisomicin
395 grain-negative
Range (; eg/ml)
41.8 31.1 31.9 50.0 77.1
12.5•`
> 200
12.5•`
>200
12.5•`
>200
12.5•`
>200
25•`>200
a Strains were regarded as susceptible , when the MICs of gentamicin, sisomicin, tobramycin micin were 6.25 ug/ml or less, and of amikacin 12.5 ug/ml or less. Table
3.
Summary
of aminoglycoside-antibiotic
No.
No. of strains
Organism
resistant
gram-negative of strains
of
MICs
and netil-
organisms.
resistant
to
Gen
Sis
Tob
Net
Ami
Escherichia coli Klebsiella spp. Serratia spp. Proteus mirabilis Proteus spp. (indole positive) Pseudomonas spp. Acinetobacter spp. and Alcaligenes spp.
114 75
4
1
0
6
3 14
1
14
0
0
43
27
24
26
5
1
0
0
7
0
Total
9 19
1
1
8
8
0 7
62
14
12
11
14
5
10
2
2
2
2
2
332
70
54
63
29
8
Strains were regarded as resistant, when the MICs of gentamicin (Gen), sisomicin (Sis), tobramycin (Tob) and netilmicin (Net) were 12.5 ,ug/ml or greater, and of amikacin (Ami) 25 leg/ml or more. Table
4.
Summary
aureus strains
of antimicrobial (Zurich,
activity
Susceptible Antibiotics
No. of strains
of five aminoglycoside
antibiotics
Geometric mean of MICs (hg/ml)
% of strains
Resistant
strainsa Range
of
MICs
No. of strains
% of strains
(ug/ml)
0.044 0.015•`1.6 0 0.026 0.007•`1.6 0 0.047 0.03•`6.25 0 Netilmicin 435 100 0.071 0.03•`1.6 0 Amikacin 433 99.4 0.422 0.1 •`6.25 2 a Strains were regarded as susceptible , when the MICs of gentamicin, micin were 6.25 ug/ml or less, and of amikacin 12.5 #g/ml or less. Gentamicin
435
100
0
Sisomicin Tobramycin
435 435
100 100
0 0
The AAC(2')
type 226) transfers
2 of ring I of aminoglycosides HK231.
It efficiently
but not amikacin capable
the acetyl group from acetyl-CoA
0 0.6
435 Staphylococcus
and inactivated In contrast,
the deoxystreptamine
gentamicin,
Geometric mean of MICs (fug/ml)
Range of MICs (frg/ml)
50
50
to the amino
sisomicin,
the aminoglycoside
strains
sisomicin, tobramycin and netil-
(see Fig. 1). This enzyme was produced
acetylated
(see Table 6).
of acetylating
against
1976).
group in position
by the Proteus tobramycin
vulgaris strain and netilmicin,
3-N-acetyltransferase27)
moiety (ring II in Fig. 1) of gentamicin
was only
and, slowly,
of
660
THE
JOURNAL
OF
ANTIBIOTICS
AUG.
s
1977
(A) The structures of the kanamycins. Positions in ring I are numbered 1'-6' (clockwise), in ring 11 1-6 (counterclockwise) and in ring III 1"-6" (counterclockwise). Position Y is the 2' position. The hydroxyl group in ring II is in position 5. Tobrarnycin is 3'deoxykanamycin B. Amikacin is I-hydroxybutyric acidkanamycin A. (B) The structures of the gentamicins. Numbering is as indicated for the kanamycins. The hydroxyl group in ring III is in position
2".
Sisomicin
is
4',5'-dehydrogentamicin C1a. Netilmicin is 1-N-acetyl-4',5'dehydrogentamicin
C1a.
(C) The structures of the neomycins. Positions are numbered as in the kanamycins.
netilmicin. amikacin
Strains
Resistance sisomicin
producing
this enzyme,
therefore,
were susceptible
to tobramycin,
netilmicin
and
to gentamicin
and
(see Table 6). to tobramycin
was recently
observed
and weak resistance
was due to the new aminoglycoside 4 of ring I and also perhaps
to amikacin,
but susceptibility
in Staph. epidermidis9) and Staph. aureus.28) 4'-nucleotidyltransferase,
adenylylating
of ring III (see Fig. l) of tobramycin,
This unusual the OH-group
amikacin
Since the gentamicins,
netilmicin
position,
reactive groups
in the corresponding
in position
and other
SCHWOTZER, F. H. KAYSERand W. SCHWOTZER, in preparation). do not contain
phenotype
strains
drugs (U.
sisomicin
and
were susceptible
to
these drugs (see Table 7). The aminoglycoside bers of Enterobacteriaceae
2"-nucleotidyltransferase29) and also in Pseudomonas
is widely observed aeruginosa.
in gentamicin-resistant
The enzyme
adenylylates
group in position 2 of ring III (see Fig. 1). Accordingly, gentamicin, sisomicin, micin were substrates for this enzyme, but amikacin was not. Strains producing were phenotypically
susceptible
to netilmicin.
The low level of resistance
mem
the hydroxyl
tobramycin and netilthis enzyme, however,
of the Pseudomonas
strain
VOL.
XXX NO.
Table
5.
8
THE
Relation
JOURNAL
OF
ANTIBIOTICS
of aminoglycoside-phosphorylating
enzymes
and inactivation
MIC (/cg/ml) Enzyme
Antibiotica
Strain
Gen Aminoglycoside 3'-phosphot ransferase type 4
Sis Tob
Staph. aureus FK 170
[APH(3')-4]
Aminoglycoside 2"-phosphotransferase
Net Ami
Staplt. aureus 5532
[APH (2")]
Inactivationb (zone diameter in tun)
Enzymenegative variant
Wild type strain 0.8 0.4
0.4 0.4
0.8
0.4
0.2 12.5
661
Neo
> 200
1.6
Gen Sis Tob
50
0.8
50 50
0.8 3.1
Net Ami
12.5 6.25
0.8 6.25
to MIC.
Extent of phosphorylation° (cpm)
Efficiency of phosphorylationd (%) 0 0 0 0 18 100
130
18/18 22/22 16/16 22/23 6/24 6/12
0.4 6.25
of antibiotics
20 0 20 6,060 9,800
6/21 6/18 6/17 6/21 18/23
100 93 27 132 12
2,500 2,800 1,600 3,200 1,500
a Abbreviations see legend of Table 3; Neo=neomycin B. b Zone diameter of assayed sample/zone diameter of control sample. A value of 6 mm means no inhibition zone around well. c Counts per minute incorporated and assayed after 2.5 hours incubation at 35°C. d The percent is relative to the phosphorylation of neomycin or gentamicin. Table
6.
Relation
of aminoglycoside-acetylating
enzymes
and
inactivation
MIC (ug/ml) Enzyme
Strain
Antibiotics
Aminoglycoside 2'-N-acctvItransferase type 2 [AAC(2')-2]
Proteus vulgaris HK231
Aminoglycoside 3-N-acetyltransferase type 2 [AAC(3)]
Proteus mirabilis HK238
Gen
50
ND
Sis
50
ND
50
ND
Tob Net
> 200
Ami
6/16 12/15 61/14 6/18 24/24
ND ND
6.25
Gen
25
3.1
Sis Tob
25
1.6
Net Ami
Inactivationb (zone diameter in mm)
Enzymenegative variant
Wild type strain
of antibiotics
3.1
0.4
0.8
0.8
1.6
3.1
to
MIC.
Extent of acetylationc (cpm)
Efficiency of acetylatio nd (%)
1,730
100 170 149 196 0
5,040 1,750 4,700 0
6/16 6/15 12/14 17/17 24/24
100 155 0 3 0
2,300 4,900 490 3,300 48
a Abbreviations see legend of Table 3. b Zone diameter of assayed sample/zone diameter of control sample. A value of 6 mm means no inhibition zone around well. c Counts per minute incorporated and assayed after 2.5 hours of incubation at 35` C. d The percent is relative to the phosphorylation of gentamicin. N D =not done. to
netilmicin
and
inherent
in
the
type
wild
this
amikacin strain.
culture
(see
Table
Variants, in
the
7)
which
degree
of
was had
resistance
due
to
lost
the to
a
mechanism
ability
other
to
netilmicin
produce and
than
enzymatic
AAD(2"),
did
modification, not
differ
from
amikacin.
Discussion
The strains
antimicrobial are
in
agreement
activity with
of other
the
five
reports16•`20).
aminoglycoside On
antibiotics the
basis
of
examined micrograms
against per
milliliter,
susceptible amikacin
662
THE
Table
7.
Relation
JOURNAL
OF
of aminoglycoside-adenylylating
ANTIBIOTICS
activity
and inactivation
MIC (,ag/ml) Enzyme
Antibiotics
Strain
Aminoglycoside 4'-nucleotidyltransferase [AAD(4')]
Gen Sis
Staph. epidermidis FK109
E. coli W677 (pHJR66)
0.1
Tob
0.05 0.05
Net Ami
0.1 12.5
0.1 0.4
Gen
25
ND
Sis Tob
25 25
ND
Pseudomonas aeruginosa HK232
is
the
1.6
ND
> 200 100
Tob Net
25
Extent of adenylylationc (cpm)
22/22 17/18 6/16 22/23 6/24
50 0 4,300 100 3,900
0 100 0 43
6/23 6/18 6/16 6/23 23/23
1,870 2,900 3,600 4,020 0
100 258 183 81 0
7/21 6/18 6/18 19/21 22/22
1,540 2, 300 2,300 1,300 0
100 830 292 109 0
0.8 0.4 0.2
6.25 3.1
to MIC.
3.1 6.25
Efficiency of adenylylationd (%)
Abbreviations see legend of Table 3. Zone diameter of assayed sample/zone diameter of control sample . Counts per minute incorporated and assayed after 2 .5 hours of incubation at 35°C. The percent is relative to the adenylylation of tobramycin or gentamicin. ND=not done.
least
may
ND ND
Gen Sis
Ami
a b C d
0.2
of antibiotics
1977
Inactivationb (zone diameter in mm)
0.05
0.02 200
Net Ami
Aminoglycoside 2"-nucleotidyltransferase [AAD(2")]
Enzymenegative variant
Wild type strain
AUG.
be
active
offset
agent,
especially
clinically
by
against
the
fact
Staphylococcus
that
amikacin
aureus.
reaches
However,
higher
levels
this
in
in
human
vitro
disadvantage
serum
than
the
other
drugs.30 Amikacin sisomicin
is and
ences
in
efficiency
frequencies the
of
activity
given.
As
resistance and
vivo
The
rate
brane
and
of
micin
not
the
micin.
the
data
of
test
In
certain
and
the
to
of
to
instance,
to
7).
no
in
vitro
strain variants,
exclusively
of
the
the
occurred,
however,
on
in
from
drug
(3). the
E.
but
the
did
not
differ
resistant
or
the
presence
or
coli
strain was
was
inhibited
could
in
the
level
susceptible
of
resistance
phenotypic of
and the
observed. to the to
to expression
aminoglycoside-modifying
sus-
netilmicin
susceptible
6.25
a
mem-
explain
susceptible by
this unknown
adenylylated
only
vitro
of
wall
was
were
highly
an
3-N-acetylated
efficiently
or in
producing
cell
7, are
that-because
assay
AAD(2")
strain
absence
cause the
the
5 •`
susceptibility
exhibits
the
microbiological the
aeruginosa
the
similar
Tables
experiments
be
through
differby
strain
Strains FK170
addition,
the
, and In
the of
would
pass A
In
producing the
with
amikacin.
explanation the
netilmicin,
by
corresponding
results
(Strain
effect.
AAC
strains
Pseudomonas
that
of
the well
the
drug.22)
simple
inactivation
isolated also
correlated
antibacterial
why
enzyme
demonstrate depend
since
understand,
The
the
an
of
however,
by
explained
strains.
phosphorylated to A
producing
followed
partly
enzymes
character
cases,
amikacin).
exert
strains
be
aminoglycoside-modifying
experiments
molecules
cytoplasm
can
aminoglycoside-resistant
other
susceptible
resistance
the
phenotypic
tube
for
activity,
hand,
not
vitro
phenotypically
difficult
examples do
in
strains,
discrepancy
among
staphylococci,
netilmicin
Enzyme-negative The
enzymes
concurrence.
of
This of
in
inactivation other
the
strains.
gentamicin-resistant
4).
substrates
phosphorylation22)-sufficient
Table
addition,
and
of were
more
(see
the
against
2
as
of
the
antimicrobial is
drugs
seen,
some
reach to
had It
strains
be
of
ceptibility
On
can
were
these
drug
Tables
enzymes
mechanism
slow
effective (see
occurrence
such
however,
intrinsic
In
the
APH(3')-4
enzyme,
still
most
of
of
of
in
the
tobramycin
netilmicin
!cg/ml this
netildrug.
of
. netil-
drug. of
bacterial enzymes.
VOL.
XXX
The
amount
and
the
8
of
such
of a
degree
given
strain
mentioned
Table 15
the
2)
all
(2')-2,
be
are
mainly
strain
to
selectively
against
these a
to
these
the
kinetic
constants
may
strains
all
can
at
play
a
contribute
to
site
of role
resistance. on
examination
the
significant
enzymes
comparative
in
resistance
occurrence
drugs
of
to
of
their
location
.
addition,
In
Thus,
the
the
an
phenotypic
wild
,
assess-
expression
type
strain
are
degree
due of
to
and
a
To
not
one
our
Five strain
the
five
and
its
strains
is
in
the
activity
may
show
AAD(2") the
AAC(3),
no
of
enzyme
known of
sisomicin. to
AAC-
rendering
frequency
susceptibility
Out
the
produced the
there
differences
antibiotics
enzymes. produced
produced
antimicrobial
gentamicin
to
nine
knowledge, The
better
to
collection,
amikacin.
sisomicin.
slightly
resistance
our
Only
sisomicin. and
strains
aminoglycoside-modifying
from
amikacin.
and only,
gram-negative
various
netilmicin
except
gentamicin
of
cultures
susceptible
the
drugs
low
by
the
gentamicin
two
with
the
663
aminoglycoside-modifying
differences
were all
resistant
inactivate
in
the
only
due
modifies
cell,
aminoglycosides
gentamicin-resistant
understandably
ANTIBIOTICS
variant. the
selected
which
strains
made
OF
bacterial
resistance by
above,
randomly
and,
of
influence
can
the
modified
enzyme-negative
As
of
of
JOURNAL
within of
mechanisms
the
corresponding
(see
enzymes activity
intrinsic
ment
THE
antibacterial
certain
of
NO.
to
resistance
Thus,
some
sisomicin.
Acknowlegements These
studies
were
supported
by
a grant
from
Schering
Corporation
.
References
1)
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PoRTHousr,
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1976
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SANTANAM,
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1973 Collaborative
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spectinomycin
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serum
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1971 of
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1973
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accurate
method
for
anti-
1966
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Specific
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1970
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226•`233,
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1966
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Laboratory testing.
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KAYSER:
YAMADA
clinical
sodium
against
The
BRZEZINSKA
of F.
ADACHI,
BRIEDIS,
H.
Antimicr.
KAYSER,
mycin
discs-corrections.
antimicrobial
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Scand.
antibiotics.
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Performance National
&
aminoglycoside 12)
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Path.
N.
nential 9)
and
laboratories.
Study. 8)
Rules
1973
Chemoth. role
factors
of sex in
of
resistance
determinants
2:
217•`223,
1972
pili
Escherichia
in
the
effective
coli.
J.
in
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1972 J.
&
H.
G.
Pseudomonas
ROBSON: aeruginosa
Comparative and
activity Enterobacterioceae.
of
netilmicin, Antimicr.
gentamicin, Agents
amikacin &
Chemoth.
and 10:
tobra592•`
664
17)
THE
Fu,
K.
P.
Agents 18)
&
&
KABINS,
S.
and
MEYER,
R.
&
MILLER, spectrum
21)
NATHAN other
L.
L.
NOVICK,
G.
P.:
for
KAYSER,
&
H.:
COURVALIN, range
C.
24)
DOWDING,
J.
26)
D.
CHEVEREAU,
strains
other
aminoglycosides.
Antimicr.
netilmicin,
In
Agents
vitro
netilmicin
a semisynthetic &
susceptibility
and
selected
Chemoth. of
derivative 10:
139•`
145,
gentamicin-resistant
aminoglycoside
of 1976
Entero-
antibiotics.
Antimicr.
&
J.
A.
N.
COHEN,
R.
proposal.
CURTIS,
Bact.
J. BIBER:
in
Biological
Rev.
Agents 111,
40:
189,
&
ST.
&
resistance
a broad-
827 •`
1976
FALKOW:
836,
Uniform
A
Letter
3:
new
63-68,
nomen-
11:
of
619624,
Staphylococcus
type
of amino-
1976
phosphotransferase
Chemoth. in
netilmicin,
10:
1976
Microbios.
Agents
gentamicin
of
Chemoth.
DATTA
aminoglycoside
Antimicr.
activity
&
3'-phosphotransferase IV:
staphylococci.
Plasmid-mediated
N.
168-
Aminoglycoside
found
of
WALTZ:
Antimicr.
broad
substrate
1977
aureus.
Antimicr.
Agents
&
1977 KAYSER
H.
P.
J.
L.
gentamicin
M.;
of
& J. BIBER:
Gentamicin
DANIELS,
13:
R.
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resistance
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GoEI'IC:
Aminoglycoside
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aureus.
aminoglycoside
J.
CABANA,
B.
Antimicr.
E.
&
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DAVIES,
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mediated
by
L.
G.
CAPMAU,
Agents
LE
modifying
the
L.
DANII.LS
&
J.
N-acetylation
resistance
2'-amino
group
WEINSTEIN:
of
BACA,
&
Chemoth.
&
Chemoth.
10:
GOEBEL,
the
H.
14:
Comparative 3:
of
258-264,
mediated
Letters
TAGGART:
P.
nucleotidylation
R-Factor FEBS
Agents
B.
plasmid-mediated
DAVIES:
antibiotics.
humans.
M.
New
&
F. enzyme
of
in
bacteria
aminoglycoside
Gentamicin
resistance
deoxystreptamine
moiety.
in Bio-
1972
MARTEL,
Antimicr. R.
& an
1974
J.
aeruginosa
BoUANCHAUD:
BENVENISTE,
DAVIES II,
598•`603,
BENVENISTE,
11 : 761 •`765, F.;
J.
acetyltransferase
Pseudomonas
LE GOFFIC,
coccus
30)
to
of
Antimicr.
antibiotic.
A &
Biochemistry
chemistry
29)
with
comparison
PASIFCZNIK:
amikacin.
50,
vitro
WEINSTEIN
ST.
DAVIES:
F. H.
M.: by
BRZEZINSKA,
D.
J.
Mechanisms
F. J.:
antibiotics.
28)
E.:
compared
1977
1976
CLOWES,
J.
AUG.
1976
mediated
27)
&
netilmicin
In
A.
enzyme
I 1 : 47 •`
BROWN, 419,
K.
M.
DEVAUD
ANTIBIOTICS
antibiotics.
677•`681,
phosphorylates
Chemoth. 25)
&
plasmids:
M.
P.
that
COHEN:
aminoglycoside
R.
of
aeruginosa
glycoside-phosphorylating 23)
S.
KRAUS
ARCIERI,
bacterial
F.
study
OF
1976
aminoglycoside
10:
H.;
R.
vitro
Pseudomonas
sernisynthetic
clature 22)
C.
Chemoth. G.
In 526•`534,
four
and
Agents
NEU: 10:
D.:
bacteriaceae
20)
C.
A.:
sisomicin, 19)
H.
Chemoth.
JOURNAL
293•`296,
483,
C.
J.
SOUSSY,
antibiotics
J.
DUVAL
in
Staphylo-
&
1976
gentamicin
resistance:
A
new
enzyme
which
modifies
1971
pharmacokinctics 478•`
CHARDON,
aminoglycoside
1973
of
BB-K8
and
kanamycin
in
dogs
and
THE JOURNAL OF ANTIBIOTICS
RESISTANCE ANTIBIOTICS:
OF BACTERIA
TO THE NEWER
AN EPIDEMIOLOGICAL
AND
655
AMINOGLYCOSIDE ENZYMATIC
STUDY
MARLYSE DEVAUD,FRITZ H. KAYSERand URSUL.A HUBER Institute of Medical Microbiology, University of Zurich CH-8028 Zurich, Switzerland (Received for publication May 8, 1977) Four hundred and thirty-five Staphylococcus aureus strains and 395 strains of gramnegative bacteria were tested for susceptibility to gentamicin, sisomicin, tobramycin, netilmicin and amikacin. None of the staphylococcal strains were resistant to the 5 drugs, except two strains that were resistant to amikacin. Eighteen percent of the gram-negative bacteria were resistant to gentamicin, 16% to tobramycin, 14% to sisomicin, 7 % to netilmicin and 2 % to amikacin. Tests from representative strains showed that these differenceswere mainly due to the production of aminoglycoside-modifyingenzymes. The lower incidence of strains resistant to sisomicin compared to gentamicin was due to a slightly better antimicrobial activity of sisomicin. Results of experiments on the efficiencyof enzymatic phosphorylation, acetylation and adenylylation, and the inactivation of aminoglycoside antibiotics were not always in concurrence with the degree of phenotypic expression of resistance. Thus, the aminoglycoside 3'-phosphotransferase of staphylococci modified amikacin, but the strains were susceptible to the drug. Similarly, the aminoglycoside 2"-nucleotidyltransferase from members of Enterobacteriaceae and Pseudomonas efficiently adenylylated and inactivated netilmicin, but the strains were susceptible to netilmicin. On the other hand, there can be factors, intrinsically inherent in some strains, contributing to their phenotypic expression of resistance level. It is inferred, therefore, that the contribution of enzymes to the degree of resistance to aminoglycoside antibiotics in a given strain can be evaluated only by comparative examination of resistance in the wild type strain and its corresponding enzyme-negativevariant. In recent years gentamicin, with or without the concomitant administration of beta-lactam antibiotics, has been used with increasing frequency in the treatment of hospital-acquired,
gram-negative
bacterial infections. This resulted in increasing emergence of gentamicin-resistant strains.
The situa-
tion is clearly demonstrated in Table 1, which contains the total number of strains examined at our institution from 1974 to 1976 and the percentage of gentamicin-resistant
isolates. Since the Institute
of Medical Microbiology in Zurich has to perform diagnostic microbiology for the University Hospital and for about 30 smaller hospitals in the area and has to serve also as a Public Health Laboratory for the Kanton of Zurich, these data can be considered to represent the prevailing situation in the eastern part of Switzerland. The frequency of 9 % of gentamicin-resistant hospitalized patients in 1976 is appallingly high.
cultures isolated from
Because resistance to gentamicin occurred mostly in strains having resistance to many other antibiotics a comparative examination of the newly developed or recently introduced aminoglycoside antibiotics seemed appropriate and necessary. This paper presents the result of examination of susceptibility of 830 gram-positive and gramnegative bacterial strains of clinical origin to five selected aminoglycoside antibiotics.
In addition,
data on susceptibility to modification of these antibiotics by enzymes isolated from representative gentamicin-resistant
strains, are presented.
These data help in explaining differences in resistance
656
THE
Table
1. Number of strains examined of gram-positive and gram-negative
JOURNAL
and percentage bacteria.
Relation to hospitalizationa
Bacteria
OF
ANTIBIOTICS
of gentamicin
AUG.
resistant
1974 (May-December) No. of strains
cultures
of clinical
1975
Genrb %
1977
isolates
1976
No. of strains
GenRb %
No. of strains
Genjtb %
stllph _vlococcus aureus
Outpatients
170
0
406
0
459
Hospitalized
pat.
1,355
0
2,685
0
2,639
Staple lococcus epidernlidis
Outpatients Hospitalized
168
1
247
0
183
pat.
1,239
1
1,899
0
1,114
02
Escherichia coli
Outpatients Hospitalized
450 2,215
0
1,130
0
0
4,742
1
1,199 4,724
0
pat.
11
0
12
0
13
0
54
0
96
5
83
6
92
0
200
1
191
4
pat.
761
6
1,886
12
1,875
18
37
0
72
0
pat.
230
5
524
8
2
0
17
13
31
pat.
31
35
145
59
529
80
0
219
0
1
1,166
1 1
239
pat.
101 548
1,154
3
7
51
61
0
5
291
383
6
Outpatients
Ci trobacte r freundii
Hospitalized
pat.
Outpatients
Klebsiella spp.
Hospitalized
Enterobucter spp.
Outpatients Hospitalized Outpatients
Serratia spp.
Hospitalized
6
0
1
2
98 571
I 9
Protcus nlirabilis
Outpatients Hospitalized
Proteus spp. indole-positive
Outpatients Hospitalized
pat.
14 150
Proteus inconstalls
Outpatients Hospitalized
2
50
3
33
pat.
15
53
24
62
27
63
57 79
0
48
0
46
0
pat.
0
68
1
71
7
Pseudonroncrs spp.
Outpatients Hospitalized
3
223
9
2,143
3 9
212
pat.
86 1,069
Further non-fermentative gram-negative rods
Outpatients Hospitalized
61 176
7
87
pat.
10
302
Total
Outpatients Hospitalized
1,249 7,921
1
2,703
91
2,813
pat.
4
15,971
6
15,712
Outpatients
Salmonella spp.
Hospitalized
a:
Status
b:
GenR=gentamicin
to gentamicin,
Bacterial
of prior hospitalization
sisomicin,
of outpatients
2 5
2
2,231
9 14
96
15
311
15
9
not determined.
resistance.
tobramycin,
netilmicin
and amikacin
Materials
and Methods
existing
in our collection
of strains.
Strains
These were isolated in 1976 from different clinical materials. They were comprised of Staphylococcus aurcus (435); Eschericlria coli (114); Klebsiella pneumoniae (71); Klebsiella ozaenae (4); Serratia marecscens (43); Elrterobaeicr cloacae (36); Enterobacter agglomerans (1); Citrobacter freundii (6); Proteus vulgaris (10); Proteus mirabilis (9); Proteus rettgeri (3); Proteus morganii (3); Proteus inconstatts (3); Salmonella spp. (15); Shigella spp. (2); Pseudomonas aeruginosa (57); Pseudomonas mcrltophilia (3); Pseudomonas spp. (2); Acinetobacter spp. (5); Alcaligenes spp. (5). All strains were identified according to standard procedures. Staph. aureus 5532 was a gentarnicin-resistant strain, isolated by PORTHOUSE et al.1) in England.
VOL. XXX NO. 8
THE JOURNAL OF ANTIBIOTICS
657
Staph. aureus S25 was a gentamicin-susceptible mutant of 5532. Both strains were provided by Dr. BROWN. E. coli W677 (pHJR66) was a gift of Dr. SMITH.2)This strain is the source of the adenylylating enzyme, widely used for enzymatic assay of gentamicin. All the rest of the strains were of wild type, isolated at our institution. Antibiotics and Chemicals. Laboratory standard preparation of gentarnicin C complex, sisomicin and netilmicin were generously supplied by Schering Corp. (Bloomfield, N.Y.); tobramycin by E. Lilly and Co. (Indianapolis, Ind.), and arnikacin by Bristol Lab. (Syracuse, N. Y.). Radio-labelled chemicals were bought from the Radiochemical Centre (Amersham, England). Dithiothreitol, ATP and acetyl-CoA were obtained from Calbiochem (Los Angeles, Calif.), and lysostaphin from Becton, Dickinson and Co. (Orangeburg, N. Y.). Inorganic salts of analytical grade were bought from Merck (Darmstadt, Germany), and agarose from Behringwerke AG (Marburg, Germany). Antibiotic Susceptibility Testing. Disc susceptibility testing was performed by the slightly modified single-disc method of BAUER et a1.3) and the U.S. Food and Drug Administration,4,5) using MUELLER-HINTON agar of Baltimore Biological Laboratories (BBL) (Cockeysville, Md.) supplemented with 5%, (v/v) old human blood from the blood bank. An inhibition zone of 15 mm or more around the 10ug gentamicin disc, for a given strain, was interpreted as being susceptible to the drug." The minimal inhibitory concentration (MIC) of drugs was determined on MUELLER-HINTON agar (BBL) according to the standard methods proposed by ERICSSON and SHERRIS." Production of Enzyme. The shock method of NossAL and HEPPEL8)was used for gram-negative organisms. The bacterial strains under study were serially subcultured on Brain Heart Infusion (BHI) agar (Difco), containing 10 pg gentamicin/ml, and inoculated into a medium composed of Bacto-tryptone (Difco), 10g; Bacto yeast extract (Difco), 50 g; NaCI, 10 g; glucose, 4 g; and an amount of water sufficient to make I liter. The pH was adjusted to 7.2. A hundred ml of the medium was inoculated with one ml of a 24-hoursold culture and incubated in a shaking water bath at 37'C until the end of logarithmic growth phase. Cells were harvested and washed twice with 40 ml of a buffer containing 0.01 M Tris-HCl and 0.03 M NaCl, pH 7.8 and were suspended in 20 ml of 0.033 M Tris-HCl buffer, pH 7.3 containing 0.003 M EDTA and 20% (w/v) sucrose. After it stood at room temperature for 10 minutes, the suspension was stirred with a magnetic stirrer for 5 minutes and centrifuged for 15 minutes at 10,000 x g and 4°C. The supernatant was discarded and the remaining sucrose was removed carefully from the wall of tubes with cotton swabs. The pellet of cells was suspended in 4 ml of an ice-cold solution of 0.0005 M MgCl2, stirred for 5 minutes and centrifuged for 15 minutes at 22,000 x g and 4oC. The supernatant (osmotic shockate) was carefully decanted to a flask and stabilized with 0.001 Mdithiothreitol. Enzymatic extracts of staphylococci were obtained either by the shock method or by lysing the cells with lysostaphin as previously described.9) Enzymatic Assays. ssays. These were similar to those described by BENVENISTE et al.10), DAVIESet al.11) and HAAS and DAVIES.12)Inactivation by adenylylation of aminoglycosides was tested in a reaction mixture consisting of 40 /Il enzymatic extract; 3 /smol of Tris-maleate buffer, pH 7.4; 1.4 /cmol MgCl2; 0.1 ,umol dithiothreitol; 40 nmol (2-3H)ATP (specific activity, 60uCi/,umol) and I nmol of drug base in a total volume of 130 lil. Inactivation by phosphorylation was tested in a reaction mixture containing 40 nmol of (y-52P)ATP (specific activity, 1 - 4 mCi/mmol). Acetylation was examined in tests comprising 5.5 nmol (1-11C)acetyl-CoA (specific activity, 36.3 mCi/mmol) and 0.4 nmol drug base. Appropriate controls were devoid of either the labelled coenzymes, or the drug base or the enzymatic extract. After incubating for 2.5 hours at 35°C, 501d were assayed for residual antibiotic by agar diffusion13) in Antibiotic Medium 5 (Difco) containing agarose instead of agar. Bacillus subtilis ATCC 6633 was used as test organism, in some cases, and the enzyme-producing strain was used in certain others. Another 50 pl were examined for the extent of adenylylation, phosphorylation or acetylation by mea-
658
THE
suring
the
and
radioactivity
incorporated
coworkers.11)
Instr.,
were
Grove,
appropriately 50ul
were
conditions
was
Ill.)
determining
which
by
Radioactivity
Downers For
JOURNAL
in
the
relative
diluted
and
assayed
ensured
10
ml
of
the the
linear
in
of
5
paper
always
described
by
spectrometer
1977
DAVIES
(Packard
scintillant. as
incubated
phosphocellulose
assay
scintillation
aminoglycosides
mixtures
were
binding
Tri-Carb
based the
AUG.
paper
a Packard
a toluene
reaction
assays
ANTIBIOTICS
phosphocellulose
counted
efficiency
by
that
the
OF
for
substrates,
2.5
binding
and
enzymatic
method
5 minutes
extracts
at
mentioned
35'C,
after
above.
These
occurring.
Elimination. Attempts
to
previously14).
obtain To
method
was
enzyme-negative
eliminate
variants
R-plasmids
of
from
Staphylococcus
strains
gram-negative
bacteria,
were the
made
sodium
as
described
dodecyl
sulfate
used.15)
Results
Antimicrobial The from
in
our
vitro
activity
of
collection
concentration
of
(MIC)
No
strain to
area
of
The
susceptibility
collection
1).
against
Shigella,
are
to
pattern
these
time,
Tables
shown.
of
strains
435
The
(S.
MITSUHASHI, The
and
data
to
contrast
ed
and
Some
strains
1976),
possessed
an
2 to
amikacin
(see
mutant
and
affinity
inhibitory
reported
by
outlined
in
was
however,
other
Table
observed do
3. to
occur
be
in
our
the
ring
III
that
of
In
tobramycin This Table
and is
the 5).
were
position
of
resistant
resistance in
still
and
addition
to
reason
for
The the
rather
I
time
Zurich
5).
(see
of
Fig. and
strain
1)
Chemotherapy
strains.22,23)
drug,
however,
APH(2")
intermediate produced
was
still
produced
MIC
of
tobramycin
is is
by
to
the and
sub-
FK170)
observed hydroxyl
acetylating
1).
amikacin.
APH(2")
resistance an
weak
(i.e.
recently the
producing
a to
amikacin was
Fig.
phosphorylat-
susceptible to
This
(see
also,
phosphorylates Strains
5532
Nomencla-
of
aminoglycosides
resistant The
are
Japan).
The
enzyme
Plasmid
epidermidis
amikacin,
moderately
The
high
the
observed.
antibiotics
the
phenotypically
unknown.
enzyme
in
Society
Staph.
ring
Table
tobramycin,
amikacin.26)
of
were
(see
APH(2"),
from
organisms,
England.1,24)
aminoglycosides.25)
taken
and 3
or
At was
aminoglycoside
Maebashi,
aureus
(see
4.
appeared
International
therefore,
Table
gentamicin
strains
modifying were
gram-negative
enzyme
APH(3')-4,
sisomicin 5).
in
staphylococcal
mechanism
Table
(see
group
in to
Enzymes of
University, Staph.
shown
aureus
abbreviations
in
of
staphylococci
of
mutant. strain
those
collection
resistant
Chemoresistance, Gunma
found
hydroxyl
gentamicin,
to
selected
minimal
as
species,
is
isolate
capable
the on
recently
staphylococci
resistance
negative
was
producing
inherent
position
low
4
by
gentamicin-resistant in
to
the
these
strains
no
Modifying
strains
commission
the
strains of
with
3'-phosphotransferases
inactivated and
The
species in
in
organisms,
2.
similar
Citrobacter
aureus
March
communication,
type
the
tests
the
personal
to
strate,
are
Staphylococcus
nomenclature
phosphorylates
In
on
from
APH(3')
enzyme
Table
bacterial
gentamicin
gentamicin-resistant
enzymatic
Group21)
strains
Staphylococcus
(January
however,
5•`7,
ture
gram-negative
in
among and
Resistance
395
summarized
found
Aminoglycoside In
against
susceptible
were Enterobacter
drugs.
Aminoglycosides
1).
of
that
isolates,
drugs
of
antibiotics
strains
examined
Table
Table
the
Salmonella, the
(see
Since
clinical
Resistant
of
resistant
fresh
of
investigators16•`20).
Activity
5 aminoglycosidic
in group
exhibited netilmicin enzyme
and with
phosphotransferaseamikacin
against
the
VOL.
XXX
Table
NO.
8
THE
JOURNAL
2. Summary of antimicrobial bacteria (Zurich, 1976).
activity
Susceptible Antibiotic
No. of strains
OF
659
of five aminoglycoside
antibiotics
strainsa
against
Resistant
Geometric mean of M ICS (,ug/ml)
% of strains
ANTIBIOTICS
Range
of
MICs
No. of strains
% of strains
70 54
17.7 13.7 15.9 7.3 2.0
325 341
82.3 86.3
0.93 0.56
0.2•`6.25
Tobramycin Netilmicin
332 366
84.1 92.7
0.80 0.84
0.1•`6.25 0.1•`6.25
63 29
Amikacin
387
98.0
1.66
0.4•`12.5
8
0.1•`6.25
strains
Geometric mean of MICs (,ug/ml)
(hg/ml)
Gentamicin Sisomicin
395 grain-negative
Range (; eg/ml)
41.8 31.1 31.9 50.0 77.1
12.5•`
> 200
12.5•`
>200
12.5•`
>200
12.5•`
>200
25•`>200
a Strains were regarded as susceptible , when the MICs of gentamicin, sisomicin, tobramycin micin were 6.25 ug/ml or less, and of amikacin 12.5 ug/ml or less. Table
3.
Summary
of aminoglycoside-antibiotic
No.
No. of strains
Organism
resistant
gram-negative of strains
of
MICs
and netil-
organisms.
resistant
to
Gen
Sis
Tob
Net
Ami
Escherichia coli Klebsiella spp. Serratia spp. Proteus mirabilis Proteus spp. (indole positive) Pseudomonas spp. Acinetobacter spp. and Alcaligenes spp.
114 75
4
1
0
6
3 14
1
14
0
0
43
27
24
26
5
1
0
0
7
0
Total
9 19
1
1
8
8
0 7
62
14
12
11
14
5
10
2
2
2
2
2
332
70
54
63
29
8
Strains were regarded as resistant, when the MICs of gentamicin (Gen), sisomicin (Sis), tobramycin (Tob) and netilmicin (Net) were 12.5 ,ug/ml or greater, and of amikacin (Ami) 25 leg/ml or more. Table
4.
Summary
aureus strains
of antimicrobial (Zurich,
activity
Susceptible Antibiotics
No. of strains
of five aminoglycoside
antibiotics
Geometric mean of MICs (hg/ml)
% of strains
Resistant
strainsa Range
of
MICs
No. of strains
% of strains
(ug/ml)
0.044 0.015•`1.6 0 0.026 0.007•`1.6 0 0.047 0.03•`6.25 0 Netilmicin 435 100 0.071 0.03•`1.6 0 Amikacin 433 99.4 0.422 0.1 •`6.25 2 a Strains were regarded as susceptible , when the MICs of gentamicin, micin were 6.25 ug/ml or less, and of amikacin 12.5 #g/ml or less. Gentamicin
435
100
0
Sisomicin Tobramycin
435 435
100 100
0 0
The AAC(2')
type 226) transfers
2 of ring I of aminoglycosides HK231.
It efficiently
but not amikacin capable
the acetyl group from acetyl-CoA
0 0.6
435 Staphylococcus
and inactivated In contrast,
the deoxystreptamine
gentamicin,
Geometric mean of MICs (fug/ml)
Range of MICs (frg/ml)
50
50
to the amino
sisomicin,
the aminoglycoside
strains
sisomicin, tobramycin and netil-
(see Fig. 1). This enzyme was produced
acetylated
(see Table 6).
of acetylating
against
1976).
group in position
by the Proteus tobramycin
vulgaris strain and netilmicin,
3-N-acetyltransferase27)
moiety (ring II in Fig. 1) of gentamicin
was only
and, slowly,
of
660
THE
JOURNAL
OF
ANTIBIOTICS
AUG.
s
1977
(A) The structures of the kanamycins. Positions in ring I are numbered 1'-6' (clockwise), in ring 11 1-6 (counterclockwise) and in ring III 1"-6" (counterclockwise). Position Y is the 2' position. The hydroxyl group in ring II is in position 5. Tobrarnycin is 3'deoxykanamycin B. Amikacin is I-hydroxybutyric acidkanamycin A. (B) The structures of the gentamicins. Numbering is as indicated for the kanamycins. The hydroxyl group in ring III is in position
2".
Sisomicin
is
4',5'-dehydrogentamicin C1a. Netilmicin is 1-N-acetyl-4',5'dehydrogentamicin
C1a.
(C) The structures of the neomycins. Positions are numbered as in the kanamycins.
netilmicin. amikacin
Strains
Resistance sisomicin
producing
this enzyme,
therefore,
were susceptible
to tobramycin,
netilmicin
and
to gentamicin
and
(see Table 6). to tobramycin
was recently
observed
and weak resistance
was due to the new aminoglycoside 4 of ring I and also perhaps
to amikacin,
but susceptibility
in Staph. epidermidis9) and Staph. aureus.28) 4'-nucleotidyltransferase,
adenylylating
of ring III (see Fig. l) of tobramycin,
This unusual the OH-group
amikacin
Since the gentamicins,
netilmicin
position,
reactive groups
in the corresponding
in position
and other
SCHWOTZER, F. H. KAYSERand W. SCHWOTZER, in preparation). do not contain
phenotype
strains
drugs (U.
sisomicin
and
were susceptible
to
these drugs (see Table 7). The aminoglycoside bers of Enterobacteriaceae
2"-nucleotidyltransferase29) and also in Pseudomonas
is widely observed aeruginosa.
in gentamicin-resistant
The enzyme
adenylylates
group in position 2 of ring III (see Fig. 1). Accordingly, gentamicin, sisomicin, micin were substrates for this enzyme, but amikacin was not. Strains producing were phenotypically
susceptible
to netilmicin.
The low level of resistance
mem
the hydroxyl
tobramycin and netilthis enzyme, however,
of the Pseudomonas
strain
VOL.
XXX NO.
Table
5.
8
THE
Relation
JOURNAL
OF
ANTIBIOTICS
of aminoglycoside-phosphorylating
enzymes
and inactivation
MIC (/cg/ml) Enzyme
Antibiotica
Strain
Gen Aminoglycoside 3'-phosphot ransferase type 4
Sis Tob
Staph. aureus FK 170
[APH(3')-4]
Aminoglycoside 2"-phosphotransferase
Net Ami
Staplt. aureus 5532
[APH (2")]
Inactivationb (zone diameter in tun)
Enzymenegative variant
Wild type strain 0.8 0.4
0.4 0.4
0.8
0.4
0.2 12.5
661
Neo
> 200
1.6
Gen Sis Tob
50
0.8
50 50
0.8 3.1
Net Ami
12.5 6.25
0.8 6.25
to MIC.
Extent of phosphorylation° (cpm)
Efficiency of phosphorylationd (%) 0 0 0 0 18 100
130
18/18 22/22 16/16 22/23 6/24 6/12
0.4 6.25
of antibiotics
20 0 20 6,060 9,800
6/21 6/18 6/17 6/21 18/23
100 93 27 132 12
2,500 2,800 1,600 3,200 1,500
a Abbreviations see legend of Table 3; Neo=neomycin B. b Zone diameter of assayed sample/zone diameter of control sample. A value of 6 mm means no inhibition zone around well. c Counts per minute incorporated and assayed after 2.5 hours incubation at 35°C. d The percent is relative to the phosphorylation of neomycin or gentamicin. Table
6.
Relation
of aminoglycoside-acetylating
enzymes
and
inactivation
MIC (ug/ml) Enzyme
Strain
Antibiotics
Aminoglycoside 2'-N-acctvItransferase type 2 [AAC(2')-2]
Proteus vulgaris HK231
Aminoglycoside 3-N-acetyltransferase type 2 [AAC(3)]
Proteus mirabilis HK238
Gen
50
ND
Sis
50
ND
50
ND
Tob Net
> 200
Ami
6/16 12/15 61/14 6/18 24/24
ND ND
6.25
Gen
25
3.1
Sis Tob
25
1.6
Net Ami
Inactivationb (zone diameter in mm)
Enzymenegative variant
Wild type strain
of antibiotics
3.1
0.4
0.8
0.8
1.6
3.1
to
MIC.
Extent of acetylationc (cpm)
Efficiency of acetylatio nd (%)
1,730
100 170 149 196 0
5,040 1,750 4,700 0
6/16 6/15 12/14 17/17 24/24
100 155 0 3 0
2,300 4,900 490 3,300 48
a Abbreviations see legend of Table 3. b Zone diameter of assayed sample/zone diameter of control sample. A value of 6 mm means no inhibition zone around well. c Counts per minute incorporated and assayed after 2.5 hours of incubation at 35` C. d The percent is relative to the phosphorylation of gentamicin. N D =not done. to
netilmicin
and
inherent
in
the
type
wild
this
amikacin strain.
culture
(see
Table
Variants, in
the
7)
which
degree
of
was had
resistance
due
to
lost
the to
a
mechanism
ability
other
to
netilmicin
produce and
than
enzymatic
AAD(2"),
did
modification, not
differ
from
amikacin.
Discussion
The strains
antimicrobial are
in
agreement
activity with
of other
the
five
reports16•`20).
aminoglycoside On
antibiotics the
basis
of
examined micrograms
against per
milliliter,
susceptible amikacin
662
THE
Table
7.
Relation
JOURNAL
OF
of aminoglycoside-adenylylating
ANTIBIOTICS
activity
and inactivation
MIC (,ag/ml) Enzyme
Antibiotics
Strain
Aminoglycoside 4'-nucleotidyltransferase [AAD(4')]
Gen Sis
Staph. epidermidis FK109
E. coli W677 (pHJR66)
0.1
Tob
0.05 0.05
Net Ami
0.1 12.5
0.1 0.4
Gen
25
ND
Sis Tob
25 25
ND
Pseudomonas aeruginosa HK232
is
the
1.6
ND
> 200 100
Tob Net
25
Extent of adenylylationc (cpm)
22/22 17/18 6/16 22/23 6/24
50 0 4,300 100 3,900
0 100 0 43
6/23 6/18 6/16 6/23 23/23
1,870 2,900 3,600 4,020 0
100 258 183 81 0
7/21 6/18 6/18 19/21 22/22
1,540 2, 300 2,300 1,300 0
100 830 292 109 0
0.8 0.4 0.2
6.25 3.1
to MIC.
3.1 6.25
Efficiency of adenylylationd (%)
Abbreviations see legend of Table 3. Zone diameter of assayed sample/zone diameter of control sample . Counts per minute incorporated and assayed after 2 .5 hours of incubation at 35°C. The percent is relative to the adenylylation of tobramycin or gentamicin. ND=not done.
least
may
ND ND
Gen Sis
Ami
a b C d
0.2
of antibiotics
1977
Inactivationb (zone diameter in mm)
0.05
0.02 200
Net Ami
Aminoglycoside 2"-nucleotidyltransferase [AAD(2")]
Enzymenegative variant
Wild type strain
AUG.
be
active
offset
agent,
especially
clinically
by
against
the
fact
Staphylococcus
that
amikacin
aureus.
reaches
However,
higher
levels
this
in
in
human
vitro
disadvantage
serum
than
the
other
drugs.30 Amikacin sisomicin
is and
ences
in
efficiency
frequencies the
of
activity
given.
As
resistance and
vivo
The
rate
brane
and
of
micin
not
the
micin.
the
data
of
test
In
certain
and
the
to
of
to
instance,
to
7).
no
in
vitro
strain variants,
exclusively
of
the
the
occurred,
however,
on
in
from
drug
(3). the
E.
but
the
did
not
differ
resistant
or
the
presence
or
coli
strain was
was
inhibited
could
in
the
level
susceptible
of
resistance
phenotypic of
and the
observed. to the to
to expression
aminoglycoside-modifying
sus-
netilmicin
susceptible
6.25
a
mem-
explain
susceptible by
this unknown
adenylylated
only
vitro
of
wall
was
were
highly
an
3-N-acetylated
efficiently
or in
producing
cell
7, are
that-because
assay
AAD(2")
strain
absence
cause the
the
5 •`
susceptibility
exhibits
the
microbiological the
aeruginosa
the
similar
Tables
experiments
be
through
differby
strain
Strains FK170
addition,
the
, and In
the of
would
pass A
In
producing the
with
amikacin.
explanation the
netilmicin,
by
corresponding
results
(Strain
effect.
AAC
strains
Pseudomonas
that
of
the well
the
drug.22)
simple
inactivation
isolated also
correlated
antibacterial
why
enzyme
demonstrate depend
since
understand,
The
the
an
of
however,
by
explained
strains.
phosphorylated to A
producing
followed
partly
enzymes
character
cases,
amikacin).
exert
strains
be
aminoglycoside-modifying
experiments
molecules
cytoplasm
can
aminoglycoside-resistant
other
susceptible
resistance
the
phenotypic
tube
for
activity,
hand,
not
vitro
phenotypically
difficult
examples do
in
strains,
discrepancy
among
staphylococci,
netilmicin
Enzyme-negative The
enzymes
concurrence.
of
This of
in
inactivation other
the
strains.
gentamicin-resistant
4).
substrates
phosphorylation22)-sufficient
Table
addition,
and
of were
more
(see
the
against
2
as
of
the
antimicrobial is
drugs
seen,
some
reach to
had It
strains
be
of
ceptibility
On
can
were
these
drug
Tables
enzymes
mechanism
slow
effective (see
occurrence
such
however,
intrinsic
In
the
APH(3')-4
enzyme,
still
most
of
of
of
in
the
tobramycin
netilmicin
!cg/ml this
netildrug.
of
. netil-
drug. of
bacterial enzymes.
VOL.
XXX
The
amount
and
the
8
of
such
of a
degree
given
strain
mentioned
Table 15
the
2)
all
(2')-2,
be
are
mainly
strain
to
selectively
against
these a
to
these
the
kinetic
constants
may
strains
all
can
at
play
a
contribute
to
site
of role
resistance. on
examination
the
significant
enzymes
comparative
in
resistance
occurrence
drugs
of
to
of
their
location
.
addition,
In
Thus,
the
the
an
phenotypic
wild
,
assess-
expression
type
strain
are
degree
due of
to
and
a
To
not
one
our
Five strain
the
five
and
its
strains
is
in
the
activity
may
show
AAD(2") the
AAC(3),
no
of
enzyme
known of
sisomicin. to
AAC-
rendering
frequency
susceptibility
Out
the
produced the
there
differences
antibiotics
enzymes. produced
produced
antimicrobial
gentamicin
to
nine
knowledge, The
better
to
collection,
amikacin.
sisomicin.
slightly
resistance
our
Only
sisomicin. and
strains
aminoglycoside-modifying
from
amikacin.
and only,
gram-negative
various
netilmicin
except
gentamicin
of
cultures
susceptible
the
drugs
low
by
the
gentamicin
two
with
the
663
aminoglycoside-modifying
differences
were all
resistant
inactivate
in
the
only
due
modifies
cell,
aminoglycosides
gentamicin-resistant
understandably
ANTIBIOTICS
variant. the
selected
which
strains
made
OF
bacterial
resistance by
above,
randomly
and,
of
influence
can
the
modified
enzyme-negative
As
of
of
JOURNAL
within of
mechanisms
the
corresponding
(see
enzymes activity
intrinsic
ment
THE
antibacterial
certain
of
NO.
to
resistance
Thus,
some
sisomicin.
Acknowlegements These
studies
were
supported
by
a grant
from
Schering
Corporation
.
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677•`681,
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M.
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1973
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BB-K8
and
kanamycin
in
dogs
and
