Research Notes 451
diagnosis of several pulmonary disorders [6], they
should be performed only after other diagnostic
procedures, e.g., bronchoalveolar lavage. In our
experience, lung biopsies are rarely performed as
the first-choice procedure for diagnosis of deep
mycosis.
PCR amplification of DNA from both mycelia
and spherules showed a single amplification
product of c. 340 bp. A BLAST search showed
homology only for Coccidioides spp. sequences,
which suggests that amplicon sequencing is
not necessary for routine diagnostic purposes.
However, further studies with more patients
are required to evaluate the sensitivity of the
present protocol. In the protocol described by
Burt et al. [7], filamentous cultures of Coccidioides
spp. were grown in broth medium and then
inactivated by heating, followed by freezing in
liquid nitrogen and lyophilisation before extrac-
tion of DNA. The present study used an alternat-
ive method for safe DNA extraction from
filamentous cultures growing on solid media.
This protocol may be performed directly on
suspicious cultures grown from clinical material,
thereby reducing the time required to obtain
definitive diagnostic results.
ACKNOWLEDGEMENTS
This work was supported by CNPq Conselho Nacional de
Desenvolvimento Cientı́fico e Tecnológico (Process:
478355 ⁄ 2003-3) and FAPESP Fundação de Amparo à Pesquisa
do Estado de São Paulo (Process: 4 ⁄ 14270-0).
REFERENCES
1. Bialek R, Kern J, Herrmann T et al. PCR assays for identi-
fication of Coccidioides posadasii based on the nucleotide
sequence of the antigen 2 ⁄ proline-rich antigen. J Clin
Microbiol 2004; 42: 778–783.
2. Cox R, Magee DM. Coccidioidomycosis: host response
and vaccine development. Clin Microbiol Rev 2004; 17: 804–
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3. Galgiani JN, Ampel NM, Blair JE et al. Coccidioidomycosis.
Clin Infect Dis 2005; 41: 1217–1223.
4. de Hoog GS, Guarro J, Gene J, Figueras MJ. Atlas of clinical
fungi. Reus: Centraalbureau voor Schimmelcultures ⁄ Uni-
versitat Rovira i Virgili, 2001.
5. Sanguinetti CJ, Dias N, Simpson AJG. Rapid silver staining
and recovery of PCR products separated on polyacrylamide
gels. Biotechniques 1994; 17: 915–918.
6. Gal AA. Use and abuse of lung biopsy. Adv Anat Pathol
2005; 12: 195–202.
7. Burt A, Carter DA, Koenig GL et al. A safe method of
extracting DNA from Coccidioides immitis. Fungal Genet
Newslett 1995; 42: 23.
 2007 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 13, 430–456
RESEARCH NOTE
The use of antibiotic-containing agars for
the isolation of extended-spectrum
b-lactamase-producing organisms in
intensive care units
G. Wilson and D. McCabe
Department of Medical Microbiology, Stirling
Royal Infirmary, Stirling, UK
ABSTRACT
MacConkey agar containing either cefotaxime
1.0 mg ⁄ L or ceftazidime 1.0 mg ⁄ L was evaluated
for use in screening for extended-spectrum
b-lactamase (ESBL)-producing organisms. The
media were evaluated using known ESBL-posit-
ive and -negative strains and 630 clinical
specimens over a 6-month period. All Entero-
bacteriaceae isolated were identified and screened
for ESBL production by phenotypic methods.
In total, 14 ESBL-producing organisms were
detected in the clinical samples. All known
ESBL-positive strains were also detected. The
use of both screening plates was required to
detect all ESBLs.
Keywords Detection, Enterobacteriaceae, extended-
spectrum b-lactamases, MacConkey selective agars,
screening media, surveillance
Original Submission: 30 November 2005; Revised Sub-
mission: 5 October 2006; Accepted: 19 October 2006
Clin Microbiol Infect 2007; 13: 451–453
10.1111/j.1469-0691.2006.01667.x
Detection of organisms producing an extended-
spectrum b-lactamase (ESBL) is not always easy
with routine susceptibility testing [1–3], and
delays in recognition, resulting in unsuitable
treatment with cephalosporins of severe infec-
tions caused by ESBL producers, has been linked
with a rise in the mortality rate [4]. There is a need
Corresponding author and reprint requests: G. Wilson,
Department of Medical Microbiology, Stirling Royal Infirmary,
Stirling, FK8 2AU, UK
E-mail: gwilson44@btinternet.com
452 Clinical Microbiology and Infection, Volume 13 Number 4, April 2007

for a sensitive and simple screening procedure to
enhance the detection of ESBL-producing bacteria
[5]. Ideally, use of a screening agar would allow
the initial isolation and recognition of potential
ESBL-producing organisms. Thus, previous
studies have attempted to develop screening
agars containing a single cephalosporin, either
cefotaxime or ceftazidime [6,7].
The present study evaluated the use of
two screening agars in combination to detect
ESBL-producing strains isolated from intensive
care unit (ICU) patients in Forth Valley Acute
Hospitals, Scotland. In total, 40 ESBL-positive
isolates, consisting of 16 strains producing known
ESBL enzyme types and 24 ESBL producers
(phenotypically determined) from clinical sam-
ples, and 50 negative controls, comprising known
b-lactamase-positive but non-ESBL-producing
clinical isolates, were used in the primary evalu-
ation of the media. In addition, Escherichia coli
ATCC25922 was used as a negative ESBL control,
and Klebsiella pneumoniae ATCC 700603 was used
as a positive ESBL control. For the clinical
evaluation, 630 routine samples were collected
from the ICUs of Forth Valley Acute Hospitals
over a 6-month period; these consisted of urines
(n = 192), tracheal aspirates (n = 122), groin
swabs (n = 200), wound swabs (n = 106) and
organisms. Isolates and controls were screened
for ESBL production by the Jarlier synergy test [8]
and a combination disk method (Oxoid). The
sensitivities and specificities (with 95% CIs) of
the two screening agars were calculated.
All 50 negative controls grew on the control
MacConkey plate, but were inhibited from grow-
ing on the two antibiotic-containing plates. The
growth of organisms producing known ESBLs is
summarised in Table 1. All of the positive con-
trols were detected by the Jarlier test. In total, 120
Table 1. Growth of organisms producing known exten-
ded-spectrum b-lactamases (ESBLs) on MacConkey agar
containing cefotaxime or ceftazidime
Mac X Mac Z
ESBL (cefotaxime 1.0 mg ⁄ L) (ceftazidime 1.0 mg ⁄ L)
SHV-2 + +
SHV-3 – +
SHV-4 – +
SHV-5 + +
SHV-6 – +
TEM-3 + +
TEM-6 + +
TEM-7 – +
TEM-8 + +
TEM-10 – +
TEM-12 – +
TEM-16 + +
TEM-17 – +
TEM-24 + +
TEM-26 + +
CTX M-15 + –

faeces (n = 10). +, presence of growth; –, absence of growth.

Mac X medium consisted of MacConkey agar
(Oxoid, Basingstoke, UK) supplemented with
Table 2. Identity and number of clinical isolates grown
cefotaxime sodium (Hoechst Marion Roussel, from clinical specimens on screening plates
Bridgewater, NJ, USA) 1.0 mg ⁄ L. Mac Z medium
Specimen type Organisms (n) ESBL-positive (n)
consisted of MacConkey agar supplemented with
ceftazidime pentahydrate (GlaxoSmithKline, Urine Escherichia coli (2)
Klebsiella oxytoca (1)
Brentford, UK) 1.0 mg ⁄ L. MacConkey agar was Enterobacter cloacae (2) 2
prepared according to the manufacturer’s instruc- Endotracheal aspirates Klebsiella pneumoniae (5) 4
Enterobacter cloacae (1)
tions; the corresponding antibiotic solutions were Pseudomonas spp. (22)
Groin swabs Escherichia coli (22)
filter-sterilised and added aseptically following Klebsiella oxytoca (5)
sterilisation of the agar. Klebsiella pneumoniae (7) 6
Hafnia alvei (1)
Each clinical sample and all controls were Stenotrophomonas maltophilia (1)
Serratia liquefaciens (1)
inoculated on a MacConkey agar control plate, Proteus mirabilis (2)
Mac X and Mac Z. All Enterobacteriaceae growing Enterobacter cloacae (1)
Enterobacter aerogenes (2)
on the control plate were identified using the Citrobacter koserii (1)
Citrobacter freundii (1) 1
API 10S system (bioMérieux, Marcy l’Etoile, Pseudomonas spp. (10)
France) and were tested for ESBL production. Wound swabs Escherichia coli (13)
Klebsiella oxytoca (7) 1
Any organism growing on the two antibiotic- Morganella morganii (1)
containing test plates was identified by the API 10S Acinetobacter baumanii (1)
Enterobacter cloacae (1)
system and the VITEK 2 system (bioMérieux). This Enterobacter aerogenes (1)
Pseudomonas spp. (3)
allowed comparisons with the test agars, as the Faeces Escherichia coli (3)
growth on the control plate reflected the overall Klebsiella oxytoca (1)
Pseudomonas spp. (2)
flora of the subject, including any ESBL-producing

 2007 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 13, 430–456

isolates from clinical specimens grew on the
screening agars (Table 2), including 14 probable
ESBL-producing organisms. Apart from two
isolates of Enterobacter cloacae, all of these pre-
sumptive ESBL producers showed enhanced
resistance to cefotaxime and ceftazidime using
the Jarlier test, and demonstrated a ‡5 mm
increase in zone diameters using the combination
disk test for the cephalosporins plus clavulanic
acid; the two Ent. cloacae isolates gave a negative
result in the combination disk test. No ESBL-
producing organisms grew only on the MacCon-
key control plate.
An initial screening test to facilitate the detec-
tion of ESBL-producing bacteria in a clinical
setting is important not only for guiding treat-
ment, but also for early implementation of appro-
priate infection control measures. In the present
study, the combined use of MacConkey screening
agars supplemented with either ceftazidime or
cefoxatime enabled 100% detection of known
ESBL producers. It is important to note that the
use of these agars is primarily to facilitate the
isolation of potential ESBL producers, and any
growth on the plates should not be taken as
definitive proof of ESBL production, which can
only be achieved by use of appropriate confirm-
atory tests. The selective plates will allow the
growth of any organism that has resistance to the
antibiotic incorporated in the agar. A recent study
has reported that this is a drawback [9], but
a compensating benefit is that the agars may
also isolate organisms that hyperproduce AmpC
enzymes, and it is clearly advantageous to know
whether ICU patients are colonised by these
organisms. Any growth on the selective agars
can also be tested for resistance mechanisms
other than ESBL production.
The ESBL-producing isolates from clinical spec-
imens that were detected in this study grew on
cefotaxime and ceftazidime concentrations of
1.0 mg ⁄ L in the Mac X and Mac Z screening
plates. Previous studies have used a range of
concentrations (0.5–2 mg ⁄ L) of cefoxatime and
ceftazidime to facilitate the isolation of ESBL-
producing organisms [6,7]. However, variations
in the efficiency of detection of ESBL-producing
organisms with a single selective antibiotic have
been shown [6], and it is therefore unlikely that a
single selective medium will detect all known
ESBL producers [10].
 2007 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 13, 430–456
Research Notes 453
In conclusion, it is suggested that two screening
plates, one containing cefotaxime 1.0 mg ⁄ L and
one containing ceftazidime 1.0 mg ⁄ L, can be used
to screen for ESBL-producing organisms. Such a
screening method would provide a quick and
easy-to-use method for detecting ESBL-producing
organisms in ICUs and elsewhere in hospitals,
and could therefore form a useful adjunct to
hospital infection control measures.
ACKNOWLEDGEMENTS
This work was presented, in part, at the 15th European
Congress of Clinical Microbiology and Infectious Disease,
Copenhagen, 2005.
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