Sixty-two antimicrobial agents, including several combinations, were examined for stability at 56°C for 30min and 121°C for 15min, respectively. A microtiter broth dilution MIC test and an agar disk diffusion test served to test each chemo-agent for residual antimicrobial activity. Eleven drugs were partially heat-labile (MICs raised four-to eight-fold after autoclaving) and 26 drugs were heat-labile (MICs raised &gt; 16-fold following autoclaving); the remainder proved heat-stable (MICs raised &lt; two-fold after autoclaving). Surprisingly, the /?-lactams, azlocillin, aztreonam, mezlocillin, and oxacillin, were remarkably heat-stable

Birgit Leonhard

Walter H Traub

CiteSeerX

Journal of Antimicrobial Chemotherapy (1995) 35, 149-154
Brief reports
Heat stability of the antimicrobial activity of sixty-two antibacterial agents
Walter H. Traub and Birgit Leonhard
Institut fur Medizinische Mikrobiologie und Hygiene, Universitat des Saarlandes,
Haus 43, D -66421 Homburg/Saar, Germany
Sixty-two antimicrobial agents, including several combinations, were examined for
stability at 56°C for 30min and 121°C for 15min, respectively. A microtiter broth
dilution MIC test and an agar disk diffusion test served to test each chemo-agent
for residual antimicrobial activity. Eleven drugs were partially heat-labile (MICs
raised four- to eight-fold after autoclaving) and 26 drugs were heat-labile (MICs
raised > 16-fold following autoclaving); the remainder proved heat-stable (MICs
raised < two-fold after autoclaving). Surprisingly, the /?-lactams, azlocillin,
aztreonam, mezlocillin, and oxacillin, were remarkably heat-stable.
Introduction
In a recent study sera from patients in early convalescence from systemic Acinetobacter
baumannii infection were tested for opsonizing antibodies against homologous isolates
(Traub, Leonhard & Bauer, 1994). Most sera contained antibacterials as determined by
a simple agar diffusion method (Traub & Leonhard, 1992) and conventional heat-
inactivation at 56°C for 30min failed to inactivate the antibacterial activity in the
majority of cases. Therefore, a systematic study was undertaken to determine the
heat-lability as opposed to heat-stability of a large number of antimicrobials.
Materials and methods
Bacterial strains
The following strains were employed: Staphylococcus aureus ATCC 25923, 5. aureus
ATCC 29213, Escherichia coli ATCC 25922, E. coli ATCC 35218, and Bacillus subtilis
ATCC 6633.
Media
Mueller-Hinton agar (MHA) and broth (MHB), brain heart infusion broth (BHIB), and
Tryptic Soy agar (TSA) were purchased from Difco Laboratories (Detroit, MI). The
bacterial strains were maintained on TSA slants at 4°C (bimonthly transfers) and, for
backup purposes, in a mixture of 1 volume of BHIB plus 1 volume of bovine serum
without additives (Behringwerke AG, Marburg, Germany) at — 65°C.
149
0305-7453/95/010149 + 06 $08.00/0 © 1995 The British Society for Antimicrobial Chemotherapy
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Table I. MICs of antibacterials after heat-treatment at 50cC for 30min or 121°C for I5min
Antimicrobial
Amikacin
Amoxycillin
Amoxycillin in 4 mg/L
clavulanic acid
Ampicillin
Ampicillin in 10 mg/L
sulbactam
Azlocillin
Aztrconam
Carbemcillin
Ccfamandole
Cefazolin
Cephalothin
Cefixime
Cefotaximc
Cefotiam
Cefoxitin
Ccfpirome
Ccftazidime
Ccflizoximc
Ccftriaxone
Ccfuroximc
Chloramphenicol
Ciprofloxactn
Clarithromycin
Clavulanic acid
Oindamycin
Coumermycin
Doxycycline
Erythromycin
Fojfomycin in 25 mg/L
G-6-P*
Fusidic acid
5. aureus ATCC
RT
05
0125
<OI25
<OI25
<0125
05
—
1
025
0-5
0-25
8
1
1
2
2
16
1
4
1
8
0125
025
16
«;0125
<0125
0 125
0-25
1
<OI25
MIC
56
05
025
<O125
<0125
CO-125
05
—
I
05
05
025
8
1
1
2
2
16
1
4
1
8
0125
0-25
16
<0 125
<0 125
0125
05
1
COI25
25923
121
05
2
2
05
05
1
—
|
128
64
32
128
128
128
5*256
Jc256
5*256
128
^256
5*256
16
025
025
J.256
025
<OI25
2
8
2
1
S aureus ATCC 29213
RT
2
1
<0 125
1
0125
2
—
4
025
05
025
16
1
05
4
2
8
2
4
1
16
0 25
025
32
0125
<OI25
025
025
1
<0 125
MIC
56
4
1
<O125
1
<OI25
2
—
4
05
05
025
16
2
1
4
2
8
2
4
1
16
025
025
32
0125
<0125
025
0-25
1
COI25
121
2
8
2
4
1
4
—
4
128
64
64
128
128
128
5»256
5*256
5*256
5*256
5*256
>256
16
0-25
025
>256
025
<0 125
4
4
2
1
E.
RT
2
4
4
4
2
8
<OI25
16
05
2
16
05
<OI25
<O125
4
<O125
CO-125
<0125
<0-125
4
4
<O125
32
32
64
4
05
32
2
5*256
coli ATC 25922
MIC
56
4
4
4
4
2
16
«;0 125
8
1
1
16
05
<OI25
<O125
4
<O125
025
<0125
<0 125
4
4
<0125
32
32
64
8
05
64
1
i256
121
2
32
64
16
8
16
025
128
5*256
5*256
5*256
8
16
J.256
5*256
64
32
8
32
>256
4
<0 125
32
J.256
128
8
4
>256
2
S256
E.
RT
1
*
4
—
16
<OI25
—
8
4
32
025
<O125
<O125
2
<0 125
<0 125
<OI25
<0 125
2
128
<0 125
32
32
32
8
05
32
1
>256
coli ATCC
MIC
56
1
—
4
—
8
<OI25
—
16
2
32
<OI25
<O125
0-25
2
<0125
<0125
<0 125
CO 125
2
64
<0 125
32
32
32
8
05
32
|
>256
35218
121
—
Jt256
—
>256
<0 125
—
>256
>256
>256
1
4
Jt256
>256
32
16
2
16
>256
128
<OI25
32
>256
64
16
16
>256
05
>256
RT
<0 125
<OI25
<OI25
C0-I25
<0125
16
—
0 25
<O-I25
<OI25
<O125
4
025
025
1
05
4
0-25
1
0-5
4
<O-I25
<O125
16
2
2
<0125
<O125
128
1
B. sublilis ATCC 6633
MIC
56
<0125
<OI25
<OI25
<OI25
<0125
16
—
0-25
<OI25
<0 125
<0125
2
1
025
05
05
4
0-25
1
0-5
4
<OI25
<OI25
32
1
1
<OI25
<OI25
128
1
121
<0-125
05
05
<0 125
<OI25
16
—
4
16
32
4
32
32
32
>2S6
>256
5*256
16
64
128
4
<0 125
<OI25
>256
4
2
025
1
128
4
4> inhibition
zones (mm)
RT
21
27
26
27
27
21
—
20
34
27
33
9
20
23
18
18
9
17
17
15
13
32
27
9
18
9
27
25
9
21
56
21
26
24
26
26
20
—
20
32
26
32
9
19
20
18
18
9
17
16
14
13
32
26
9
18
9
27
25
9
20
121
21
19
17
22
23
18
—
22
9
9
9
9
9
9
9
9
9
9
9
9
12
32
26
9
4
9
17
17
9
9
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Gentamicin
Imipenem
Jojamycin
Kanamycin
Lalamoxcf
Meropenem
Mcthicillin
Mczlocillm
Mupirocin
Nalidixk acid
Nclilmicin
Nitrofurantoin
Norfloxacin
Novobiocin
Ofloxaan
Oxacillin
Penicillin G
Piperacillin
Pipcracillin +
tazobactam 8:1
Polymyxin B
Rifampicin
Sulbaclam
Tazobactam
Teicoplanin
Tcmocillin
Tctracycline
Ticarcillin
Ticarcillin in 4 mg/L
clavulank acid
Tobramycin
Trimethoprim
Co-lrimoxazole
Vancomycinm
<OI25
<0125
2
05
8
<OI25
2
|
025
64
<OI25
8
1
025
025
025
<OI25
|
05
32
<OI25
128
64
1
>256
05
|
05
0125
1
006/
1 2
1
<O125
<O125
2
05
8
<OI25
2
1
025
64
<OI25
4
1
0-5
025
025
<OI25
I
|
32
<OI25
128
64
05
>256
05
1
05
0-125
1
012/
2-38
1
<0 125
!>256
4
1
>256
2
8
2
05
64
<OI25
64
1
05
025
05
025
16
16
128
<0125
>256
128
1
>256
32
05
05
0 125
1
012/
2 38
2
0-25
<OI25
2
1
8
<O125
1
4
025
64
025
8
1
025
05
025
2
2
1
32
<OI25
64
025
_
025
4
05
025
2
05/
9-5
05
025
<O125
2
1
8
<0125
2
2
0-25
64
025
8
1
05
05
025
2
2
1
32
<0125
—
64
025
—
025
4
1
025
2
05/
9-5
05
025
>256
2
1
>256
2
32
8
05
64
025
32
1
05
05
05
16
32
16
64
<OI25
—
128
2
—
32
8
1
025
2
05/
9-5
1
025
0 25
—
2
<OI25
<O125
4
128
2
05
8
<0-125
128
<0125
—
2
2
<0 125
4
64
16
05
8
4
025
1
0 12/
2-38
—
05
05
—
2
<O125
<0 125
—
4
128
2
025
8
<OI25
128
<OI25
—
2
2
<O125
8
32
—
32
05
8
8
1
1
012/
2 38
—
05
>256
—
4
5>256
0 25
—
8
128
2
05
32
<OI25
128
<OI25
—
64
32
025
16
64
5*256
32
128
128
1
1
012/
2 38
—
1
025
—
2
<O125
<O125
—
64
2
025
8
<0 125
64
<OI25
—
—
—
4
<OI25
4
32
128
—
4
05
>256
8
05
1
05/
9-5
—
05
025
—
4
<0 125
<0 125
—
—
128
2
05
16
<0 125
64
<O125
—
—
8
<O125
4
32
128
—
8
05
Je256
16
05
1
05/
95
—
05
>256
—
2
>256
025
128
2
05
32
<0125
128
<OI25
—
32
05
8
64
>256
J»256
64
>256
>256
05
1
05/
95
—
<OI25
<OI25
025
<OI25
4
<O125
<OI25
2
<OI25
2
<0125
8
<0125
1
<OI25
<OI25
4
05
05
4
<0125
64
16
<0 125
—
<0125
0 25
<0125
<0125
<0125
<0-03/
0-6
025
<OI25
<OI25
025
<OI25
4
<OI25
<OI25
2
<0 125
2
<0 125
16
<0 125
1
<0 125
<0125
8
1
05
4
<OI25
32
16
<OI25
—
<OI25
025
<0125
<OI25
< 0 125
006/
1-2
025
<OI25
>256
05
<O125
>256
1
1
4
025
2
<OI25
34
<OI25
2
<OI25
025
16
16
2
4
<O125
64
64
05
—
32
2
<0 125
«;o 125
<0125
KOOil
06
025
24
32
20
22
10
27
26
22
23
14
24
9
28
17
28
24
32
21
21
9
23
9
9
15
—
25
23
23
25
29
29
17
24
31
20
22
10
27
25
21
23
14
24
9
27
17
28
24
32
20
20
9
23
9
9
15
—
25
23
23
25
29
29
17
24
9
19
22
9
16
14
17
21
14
24
9
26
15
27
21
26
11
II
9
21
9
9
II
—
9
23
23
24
29
29
14
•G-6-P = glucose-6-phosphatc.
*—, data not prejenlcd as strains were highly resistant.
RT. No treatment; 56 - 56°C for 30 min; 121 - I2I°C for 15 min.
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152 W. H. Traub and B. Leonhard
Antimicrobials
Reference substances of the antimicrobials listed in Table I were gifts of the respective
manufacturers; stock solutions were prepared according to the manufacturers' specifi-
cations to a concentration of 2560 mg/L (co-trimoxazole contained 320 mg/L trimetho-
prim and 6080 mg/L sulphamethoxazole), sterilized by membrane nitration (0-22 /im;
Nalge Sybron Corp., Rochester, NY), dispensed as 1-5 mL aliquots, and stored at
-65°C.
Heat-exposure of antimicrobials
The stock solution of each antimicrobial drug was diluted five-fold in MHB (512 mg/L)
except for co-trimoxazole which was 64/1216 mg/L (trimethoprim/sulphamethoxazole).
This solution was divided into three aliquots. The first aliquot was held at room
temperature, the second aliquot was exposed to 56°C for 30 min in a waterbath, and
the third aliquot was autoclaved at 121°C for 15 min.
Antimicrobial susceptibility tests
A previously described microtiter method (Traub, Spohr & Bauer, 1986), using MHB
and bacterial inoculum adjusted to 1-5 x 105cfu in 01 mL, was used to determine the
MICs of each treated or untreated solution (RT, 56°C and 12 PC) against the five test
strains. The concentrations tested ranged from 128 to 0-125 mg/L (co-trimoxazole
32/608-0.06/1-2 mg/L (trimethoprim/sulphamethoxazole)). The microtiter plates were
incubated at 35°C overnight. In addition the treated and untreated solutions were
further diluted two-fold, and tested for residual activity against B. subtilis strain ATCC
6633 using a simple agar diffusion procedure (Traub & Leonhard, 1992); sterile disks
(9 mm diameter) (Schleicher & Schiill, Dassel, Germany) were charged with 10 nL of
the respective antibiotic solution and pressed gently against the surface of B. subtilis -
inoculated MHA plates (14 cm diameter) which were incubated at 35°C overnight. The
diameters of resultant inhibition zones were recorded in mm (9 mm = No zone of
activity).
Results and discussion
Among the 62 individual antimicrobials or combinations tested, the majority of
/Mactam antibiotics were extensively or totally inactivated by autoclaving (Table I).
Surprisingly, however, azlocillin, aztreonam, mezlocillin, and oxacillin were remarkably
heat-stable. All aminoglycosides, all quinolones, chloramphenicol, clindamycin,
coumermycin, fosfomycin, josamycin, mupirocin, novobiocin, co-trimoxazole,
trimethoprim, and vancomycin proved heat-stable. Several /?-lactam antibiotics, fusidic
acid, nitrofurantoin, polymyxin B, rifampicin, and teicoplanin were partially heat-labile,
in that antibacterial activity was reduced four- to eight-fold. Erythromycin, doxycycline,
and tetracycline were markedly heat-labile. Table II summarizes these findings and
groups the antimicrobials on the basis of their heat stability.
To the best of our knowledge, this is the first comprehensive examination of a large
number of antimicrobials with regard to heat-lability. The data obtained might simplify
the development and preparation of antibiotic-containing selection media, in that
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Table II. Heat-stability of 62 antimicrobials or combinations, after autoclaving at 121 °C, 15min
Heat-stable
(MICs raised < two-fold)
(n = 25)
Partially heat-stable
(MICs raised four- to eight-fold)
Heat-labile
(MICs raised ^16-fold)
(n = 26)
Amikacin
Azlocillin
Aztreonam
Clarithromycin
Chloramphenicol
Ciprofloxacin
Clindamycin
Coumermycin
Fosfomycin
Gentamicin
Josamycin
Kanamycin
Mezlocillin
Mupirocin
Nalidixic acid
Netilmicin
Norfloxacin
Novobiocin
Ofloxacin
Oxacillin
Sulbactam
Tobramycin
Trimethoprim
Trimethoprim +
Sulphamethoxazole
Vancomycin
Amoxycillin
Ampicillin
Ampicillin + sulbactam
Carbenicillin
Fusidic Acid
Nitrofurantoin
Penicillin G
Polymixin B
Rifampicin
Tazobactam
Teicoplanin
Amoxycillin + clavulanic acid
Cefamandole
Cefazolin
Cephalothin
Cefixime
Cefotaxime
Cefotiam
Cefoxitin
Cefpirome
Ceftazidime
Ceftizoxime
Ceftriaxone
Cefuroxime
Clavulanic acid
Doxycycline
Erythromycin
Imipenem
Latamoxef
Meropenem
Methicillin
Piperacillin
Piperacillin + tazobactam
Temocillin
Tetracycline
Ticarcillin
Timentin
sa
ss
£
o
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ED
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154 W. H. Traub and B. Leonhard
candidate heat-stable antibiotics may be added to rehydrated media before autoclaving.
An additional noteworthy finding was that exposure to 56°C for 30 min failed to raise
the MICs of any of the agents tested by more than two-fold (the only exception was
tobramycin assayed against E. coli ATCC 25922). The disc diffusion test results on the
whole agreed with the MIC results. However, since MIC tests are relatively insensitive
to small changes in concentration heat treatment at 56°C for 15 min could significantly
reduce concentrations measured by a more sensitive technique such as immunoassay,
HPLC or bioassay. These data indicate that the majority of the tested antimicrobials
would not be destroyed by autoclaving prior to disposal, a potentially ecologically
significant finding, although since only antimicrobial activity was measured it cannot
be ruled out that degradation or conversion to other microbiologically active products
did not take place.
Acknowledgements
The gifts of antimicrobials are appreciated. We thank Ms. Beate Ebel for the
preparation of this manuscript.
References
Traub, W. H. & Leonhard, B. (1992). Detection of antimicrobial drugs with Bacillus subtilis strain
ATCC 6633: an update. Chemotherapy 38, 155-8.
Traub, W. H., Leonhard, B. & Bauer, D. (1994). Exposure of clinical isolates of Acinetobacter
baumannii and genospecies 3 to defibrinated human blood with and without added human
'natural' or (patient) immune antibodies. Chemotherapy, in press.
Traub, W. H., Spohr, M. & Bauer, D. (1986). Streptococcus faecalis: in vitro susceptibility to
antimicrobial drugs, single and combined, with and without defibrinated human blood.
Chemotherapy 32, 270-85.
(Received 8 July 1994; accepted 29 September 1994)
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Brief reports Heat stability of the antimicrobial activity of sixty-two antibacterial agents

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Brief reports Heat stability of the antimicrobial activity of sixty-two antibacterial agents

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