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Correspondence
2. Bush, K. & Jacoby, G. A. (1997). Nomenclature of TEM
/8-lactamases. Journal of Antimicrobial Chemotherapy 39,1-3.
3. Sirot, D., Recule, C, Chaibi, E. B., Bret, L., Croize, J., ChanalClaris, C. ef al. (1997). A complex mutant of TEM-1 /3-lactamase
with mutations encountered in both IRT-4 and extended-spectrum
TEM-15, produced by an Escherichia coli clinical isolate. Antimicrobial Agents and Chemotherapy 41,1322-5.
4. Nordmann, P. & Naas, T. (1994). Sequence analysis of PER-1
extended-spectrum /^-lactamase from Pseudomonas aeruginosa
and comparison with class A /^-lactamases. Antimicrobial Agents
and Chemotherapy 38,104-114.
5. Vahaboglu, H., Ozturk, R., Aygun, G., Coskunkan, F., Yaman,
A., Kaygusuz, A. et al. (1997). Widespread detection of PER-1-type
extended-spectrum /3-lactamases among nosocomal Acinetobacter
and Pseudomonas aeruginosa isolates in Turkey: a nationwide
-multicenter study. Antimicrobial Agents and Chemotherapy 41,
2265-9.
6. Kunkel, T. A., Roberts, J. D. & Zakour, R. A. (1987). Rapid and
efficient site-specific mutagenesis without phenotypic selection.
Methods in Enzymology\5A, 367-82.
7. Bouthors, A. T., Dagoneau-Blanchard, N., Naas, T., Nordmann,
P., Jarlier, V. & Sougakoff, W. (1998). Role of residues 104, 164,
166, 238 and 240 in the substrate profile of PER-1 /3-lactamase
hydrolysing third-generation cephalosporins. Biochemical Journal
330,1443-9.
8. Bush, K., Macalintal, C, Rasmussen, B. A., Lee, V. J. & Yang, Y.
(1993). Kinetic interactions of tazobactam with /^-lactamases from
all major structural classes. Antimicrobial Agents and Chemotherapy 37, 851-8.
Characterization of the extended-spectrum
/^-lactamases and determination of the antibiotic
susceptibilities of Klebsiella pneumoniae
isolates in Hungary
JAntimicrob Chemother 1998; 42:401-403
Zoltan Pragai, Zsofia Koczian and Elisabeth Nagy*
Department of Clinical Microbiology, Albert SzentGybrgyi Medical University, Somogyi Bela ter 1,
H-6701, PO Box 482, Szeged, Hungary
*Corresponding author. Tel/Fax: +36-62-310-981.
Sir,
Transferable ^-lactamases which confer resistance to
extended-spectrum cephalosporins are a common mechanism of resistance in aerobic Gram-negative bacilli,
particularly Klebsiella pneumoniae} Of these enzymes,
SHV-type extended-spectrum ^-lactamases (ESBLs)
confer a greater level of resistance than TEM-type (5lactamases, even to /Mactam//Mactamase inhibitor combinations, thereby confounding efforts to effectively treat
patients with infections caused by these bacteria. 2 In most
European countries and other parts of the world, there are
published data for the types and prevalences of ESBLs in
K. pneumoniae isolates. However, to date, no such data
have been available in Hungary. The aims of the present
study were to collect these data and to evaluate the
activities of a wide range of antibiotics against K.
pneumoniae isolates.
Altogether, 170 non-replicate K. pneumoniae clinical
isolates were collected during 1996 by laboratories in
five regions of the country (eight strains from western
Hungary, ten from the north-west, 27 from northern
Hungary, 11 from Budapest and 114 from Szeged). All of
the strains isolated locally were identified according to
standard laboratory techniques and those referred from
other laboratories were identified by the ATB system
(bioMerieux, Marcy l'Etoile, France). The antibiotics
tested were as follows: amoxycillin, cefoxitin, cefuroxime,
ceftazidime, cefotaxime, cefepime, cefpirome, aztreonam,
imipenem, ampicillin/sulbactam (in a ratio of 2:1), coamoxiclav (amoxycillinxlavulanic acid, 2:1), piperacillin/
tazobactam (tazobactam at a fixed concentration of
4 mg/L), ceftazidime/clavulanic acid (clavulanic acid at a
fixed concentration of 4 mg/L), ciprofloxacin, gentamicin,
and amikacin. A double-disc diffusion test (DDDT), with
co-amoxiclav, ceftazidime and cefotaxime, was used to
detect ESBL production and the Etest ESBL strip (AB
Biodisk, Solna, Sweden) was used to confirm the results of
the DDDT. MICs were determined by the Etest on
Mueller- Hinton agar according to the manufacturer's
instructions; susceptibility categories were assigned in
accordance with recommendations of the National
Committee for Clinical Laboratory Standards. 3
For the DNA analyses, plasmid DNA was extracted
with a DNA purification kit (Promega, Madison, WI,
USA) and used as a template for the amplification of the
SHV gene with SHV-specific primers as described
previously.4 The amplification product was digested with
Pstl (Pharmacia, Uppsala, Sweden) and separated on a
polyacrylamide gel with known SHV standards; singlestranded DNA was stained with silver nitrate as described
previously.4 Escherichia coli control strains harbouring the
SHV-type genes were kindly provided by AB Biodisk.
For the /^-lactamase assays, the strains were grown in
Mueller-Hinton broth, the cells disrupted by sonication
and the crude enzyme in the supernatants quantified
spectrophotometrically with nitrocefin as the substrate, as
described previously.5 The protein contents of the samples
were determined by the method of Lowry with bovine
serum albumin as the standard. 6
Fifteen (9%) of the 170 K. pneumoniae isolates
produced ESBLs, all of which were of the SHV type (12
SHV-2 /3-lactamases and three SHV-5). Nine strains were
isolated in Szeged, three in Budapest, two in northern
Hungary and one in western Hungary. The activities of the
16 antibiotics against the 15 strains are shown in the Table.
All of the isolates were resistant to amoxycillin and
401
2
3
4
5
6
7
8
9
10
11
12
13
14
15
>256 :>256 :>256 >256 :>256 :>256 :>256 :>256 >256 :>256 :>256 :>256 :>256 :>256 :>256
64
4
64
64
8
4
4
8
16
4
4
4
8
4
8
64
32
64
64
64
256
64
64
128
32
256
256
128
32
256
4
8
32
64
16
4
8
4
8
16
8
32
16
64
16
4
4
8
4
8
32
16
8
4
8
8
16
32
2
32
2
1
1
2
2
2
2
1
1
1
1
4
4
2
0.5
4
4
2
4
2
4
4
4
4
4
2
8
8
2
16
2
4
32
4
1
1
2
1
2
8
4
16
16
32
4
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
256
256
256
256
32
128
256
256
256
256
256
256
256
16
256
4
4
4
4
4
32
8
4
8
4
32
8
2
8
64
4
64
4
256
16
128
32
4
256
32
128
16
8
256
256
0.25
0.5
0.12
0.12
1
0.25
1
0.25
1
0.25
1
1
1
1
2
0.12
0.12
1
0.5
0.12
0.12
0.25
0.25
0.12
0.12
0.12
0.12 >32
0.12
0.12
8
8
64
4
64
4
64
128
16
8
8
32
8
8
128
1
1
32
1
1
2
2
2
2
16
1
0.12
2
1
4
60
62
54
48
243
328
640
1
"Strains 1-12 produced SHV-2/J-lactamases and strains 13-15 produced SHV-5 /^-lactamases.
Amoxycillin
Cefoxitin
Cefuroxime
Ceftazidime
Cefotaxime
Cefepime
Cefpirome
Aztreonam
Imipenem
Ampicillin/sulbactam
Co-amoxiclav
Piperacillin/tazobactam
Ceftazidime/clavulanate
Ciprofloxacin
Gentamicin
Amikacin
/^-Lactamase activity
(U/mg of protein)
Antibiotic
MIC (mg/L)
Table. In-vitro activities of various antibiotics for 15 ESBL-producing K. pneumoniae strains" isolated in Hungary
Correspondence
cefuroxime, while 12 were susceptible to cefoxitin. The
MICs of cefotaxime, ceftazidime and aztreonam were
variable, but the MICs of ceftazidime and aztreonam for
strains producing SHV-5 /3-lactamases were, in general,
higher than those for strains producing SHV-2 enzymes,
whereas the converse applied to cefotaxime. Cefepime
and cefpirome were more active than the third-generation
cephalosporins, reflecting their low affinities for plasmidmediated /3-lactamases and their ability to resist hydrolysis
by these enzymes. Cefpirome, but not cefepime, was less
active against strain no. 9 which exhibited greater (5lactamase activity than the other strains. Imipenem was
highly active against all of the strains and piperacillin/
tazobactam was more active against SHV-5-producing
isolates than those producing SHV-2, an observation that
has been made previously by Jacoby & Carreras.2 Most of
the isolates were resistant to ampicillin/sulbactam. On the
other hand, all but three were susceptible to co-amoxiclav;
moreover, the ^-lactamase activities of the three resistant
strains, all of which expressed SHV-2 /3-lactamases, were
up to 13-fold greater than those of susceptible strains. In
the presence of clavulanic acid, the MICs of ceftazidime
for all 15 strains were markedly reduced and even the
resistant strains became susceptible. Thirteen of the
strains were resistant to gentamicin and one exhibited
resistance and a second reduced susceptibility to amikacin.
All but one strain were susceptible to ciprofloxacin.
In the present study, the ESBLs expressed by
Hungarian isolates of K. pneumoniae were shown to be
either SHV-2 or SHV-5—a pattern that is consistent with
those reported by investigators in many other countries.1
Despite cefepime and cefpirome having been shown to be
more active in vitro against the isolates than the other
/Mactams tested, with the exception of imipenem, these
agents should be used with caution as treatment of
patients with infections caused by ESBL-producing
strains, particularly when there are large numbers of
bacteria (producing corresponding large amounts of
/3-lactamases) at the sites of infection.7 More than 50% of
strains producing SHV-2 ^-lactamases, but none of those
producing SHV-5 enzymes, exhibited resistance to
piperacillin/tazobactam, while only a minority of the SHV2 producers were resistant to co-amoxiclav. Jacoby &
Carreras2 observed that the /^-lactamase activities were
significantly lower in SHV derivatives, particularly SHV-5,
than in SHV-1-producing strains, presumably because the
substitutions that broaden the spectra of SHV-1 /?lactamases at the same time lower the catalytic efficiency
of extended-spectrum enzymes.
In conclusion, this study has shown that K. pneumoniae
isolates in Hungary produce SHV-type ESBLs exclusively
and are often resistant to multiple antibiotics, including
non-/Mactams.
References
1. Jacoby, G. A. & Medeiros, A. A. (1991). More extendedspectrum /3-lactamases. Antimicrobial Agents and Chemotherapy
34,1697-704.
2. Jacoby, G. A. & Carreras, I. (1990). Activities of /3-lactam
antibiotics against Escherichia coli strains producing extendedspectrum /3-lactamases. Antimicrobial Agents and Chemotherapy
34, 858-62.
3. National Committee for Clinical Laboratory Standards. (1995).
Performance Standards for Antimicrobial Susceptibility Testing:
Sixth Informational Supplement, Document M100-S6. NCCLS,
Wayne, PA.
4. M'Zali, F. H., Gascoyne-Binzi, D. M., Heritage, J. & Hawkey, P.
M. (1996). Detection of mutations conferring extended-spectrum
activity on SHV /^-lactamases using polymerase chain reaction
single strand conformational polymorphism (PCR-SSCP). Journal
of Antimicrobial Chemotherapy 37, 797-802.
5. O'Callaghan, C. H., Morris, A., Kirby, S. M. & Shingler, A. H.
(1972). Novel method for detection of ^-lactamases by using a
chromogenic cephalosporin substrate. Antimicrobial Agents and
Chemotherapy 1,283-8.
6. Lowry, O. H., Rosebrough, N. J., Farr, A. G. & Randall, R. J.
(1951). Protein measurement with the Folin phenol reagent.
Journal of Biological Chemistry 193, 265-75.
7. Rice, L. B., Yao, J. D. C, Klimm, K., Eliopoulos, G. M. &
Moellering, R. C. (1991). Efficacy of different /3-lactams against an
extended-spectrum /3-lactamase-producing Klebsiella pneumoniae
in the rat intra-abdominal abscess model. Antimicrobial Agents and
Chemotherapy 35,1243-4.
Comparison of the 5 u£ disc and the NeoSensitab for determining the susceptibilities of
Staphylococcus aureus isolates to mupirocin
J Antimicrob Chemother 1998; 42:403-405
J. E. Finlay*, L. A. Miller and J. A. Poupard
SmithKline Beecham Pharmaceuticals, 1250 South
Collegeville Road, Collegeville, PA 19426, USA
*Tel: +1-610-917-6275; Fax: +1-610-917-4617.
Sir,
Mupirocin is a topical antibiotic that has excellent in-vitro
activity against staphylococci (both methicillin-susceptible
and -resistant) and streptococci. It is currently available in
three formulations: a polyethylene glycol-based ointment
for treating skin infections; a soft paraffin base for the
eradication of Staphylococcus aureus nasal colonization;
and a cream formulation that has recently been licensed in
the USA for the treatment of patients with infected
traumatic skin lesions.
The susceptibilities of clinical isolates to mupirocin can
be determined by a variety of methods, including disc
403