ABSTRACTThis study describes the development of a method for rapid preliminary species identification of bacteria from positive blood culture vials. The method yielded preliminary identification results for 496 (92%) of 541 positive blood cultures within 5 h. The method was capable of identifying the most frequently isolated bacteria (i.e., Staphylococcus aureus, coagulase-negative staphylococci, Escherichia coli, Streptococcus pneumoniae and Enterococcus spp.) to the species level. The method can be established easily, with a materials cost of 2–5 Euros per sample

Lundberg, C.

Tegmark-Wisell, K.

Wretlind, B.

Özenci, V.

Elsevier - Publisher Connector 

the treatment of mild to moderate scrub typhus. Antimicrob
Agents Chemother 2007; 51: 2011–2015.
6. Goddard J. Chigger mites and scrub typhus. Infect Med 2000;
17: 236–239.
7. Gentry JW, Yueh CS, Wah PO. Preliminary observations on
Leptotrombidium (Leptotrombidium) akamushi and Leptotreom-
bidium (Leptotrombidium) deliensis in their natural habitat in
Malaya: (Acarina, Trombiculidae). Am J Hyg 1963; 78: 181–
190.
8. Yi KS, Chong Y, Covington SC et al. Scrub typhus in Korea:
importance of early clinical diagnosis in this newly recog-
nized endemic area. Mil Med 1993; 158: 269–273.
9. Strickman D, Tanskul P, Eamsila C, Kelly DJ. Prevalence of
antibodies to rickettsiae in the human population of
suburban Bangkok. Am J Trop Med Hyg 1994; 51: 149–
153.
RESEARCH NOTE
Rapid culture and identification: a practical
method for early preliminary laboratory
diagnosis of sepsis
V. O¨zenci, K. Tegmark-Wisell, C. Lundberg
and B. Wretlind
Division of Clinical Microbiology, Karolinska
Institutet, Karolinska University Hospital,
Huddinge, Stockholm, Sweden
ABSTRACT
This study describes the development of a
method for rapid preliminary species identifica-
tion of bacteria from positive blood culture vials.
The method yielded preliminary identification
results for 496 (92%) of 541 positive blood
cultures within 5 h. The method was capable of
identifying the most frequently isolated bacteria
(i.e., Staphylococcus aureus, coagulase-negative
staphylococci, Escherichia coli, Streptococcus pneu-
moniae and Enterococcus spp.) to the species level.
The method can be established easily, with a
materials cost of 2–5 Euros per sample.
Keywords Blood cultures, diagnosis, identification,
rapid, sepsis, spot tests
Original Submission: 19April 2007; Revised Submis-
sion: 8 August 2007; Accepted: 27 September 2007
Clin Microbiol Infect 2008; 14: 177–180
10.1111/j.1469-0691.2007.01897.x
Early initiation of adequate antibiotic treatment in
cases of sepsis significantly reduces mortality,
morbidity and hospital costs [1]. Rapid species
identification could help to correct inadequate
empirical antimicrobial treatment. Rapid molecu-
lar-based species identification methods have
been developed, but these are expensive, are
labour-intensive and require special equipment
[2,3]. The present study describes the develop-
ment of a method for rapid preliminary species
identification from positive blood culture vials.
Significant growth of bacteria from blood culture
vials was obtained within a few hours on agar
plates.
Data were collected prospectively at Karolinska
University Hospital, Huddinge, Sweden, during
6 months in 2006. In total, 541 positive blood
cultures were included in the study, with only
one culture for each patient. The method was also
tested using nine samples from sterile body sites
other than blood. The Bactec 9240 automated
blood culture system with both aerobic and
anaerobic vials (Becton Dickinson, Sparks, MD,
USA) was used in the study. The results were
compared with those obtained by conventional
microbiological methods.
In preliminary experiments to determine the
optimal incubation time and amount of inoculum
for rapid culture, reference strains of Escherichia
coli and Staphylococcus aureus, as well as eight
clinical samples, were inoculated in blood culture
vials together with 5 mL of horse blood. When the
automated blood culture system signalled
growth, samples of the blood culture were anal-
ysed. The results revealed that five drops yielded
optimal growth and provided the best discrimi-
nation of single colonies after a 4-h incubation
period.
Accordingly, five drops of blood culture
(n = 541) were then subcultured in duplicate on
blood agar, chocolate blood agar with haematin,
and cysteine lactose electrolyte-deficient agar,
followed by incubation at 37C for 4 h in both
an aerobic and a CO2 5% v ⁄ v atmosphere. When
anaerobic bacteria were suspected, similar
amounts of culture were subcultured on brucella
Corresponding author and reprint requests: V. O¨zenci, Karo-
linska University Hospital, Clinical Microbiology, F72, Hudd-
inge, 141 86 Stockholm, Sweden
E-mail: volkan.ozenci@karolinska.se
Research Notes 177
 2007 The Authors
Journal Compilation  2007 European Society of Clinical Microbiology and Infectious Diseases, CMI, 14, 174–189
agar plates and incubated anaerobically at 37C
for 4 h.
Preliminary identification of the infecting path-
ogen was performed using a panel of validated
desktop spot tests, including catalase, oxidase,
indole spot and L-pyrrolidonyl-b-naphthylamide
(Remel Inc., Lenexa, KN, USA) tests, agglutina-
tion tests for Staph. aureus (Staphaurex latex test;
Remel Europe Ltd, Dartford, UK), group A, B, C,
D and G streptococci, Streptococcus pneumoniae
(Oxoid, Basingstoke, UK) and Salmonella spp.
(Reagensia, Stockholm, Sweden), and antibiotic
disk tests, including nitrocefin (Biodisk, Solna,
Sweden), optochin (ethyl hydrocupreine), vanco-
mycin, aztreonam, linezolid, ampicillin and met-
ronidazole (Oxoid). In the last phase of the
study, 4-nitrophenyl-b-D-glucopyranosiduronic
acid plates were used in combination with a
positive indole spot test for the preliminary
diagnosis of E. coli [4]. Ampicillin disks were
used to detect ampicillin-resistant enterococci, and
nitrocefin disks were used to detect b-lactamase-
producing Haemophilus spp.
Fig. 1 shows the algorithm that was developed
to guide the preliminary identification of bacteria
following rapid culture. Depending on the result
Fig. 1. Algorithm for the preli-
minary identification of bacteria
following rapid culture. *Lactose-
fermenting organisms changed the
colour of the cysteine lactose electro-
lyte-deficient (CLED) agar from
green to yellow. GAS, group A strep-
tococci; GBS, group B streptococci;
GCS, group C streptococci; GGS,
group G streptococci; Aggl., aggluti-
nation; CoNS, coagulase-negative
staphylococci; PGUA, 4-nitrophe-
nyl-b-D-glucopyranosiduronic acid.
178 Clinical Microbiology and Infection, Volume 14 Number 2, February 2008
 2007 The Authors
Journal Compilation  2007 European Society of Clinical Microbiology and Infectious Diseases, CMI, 14, 174–189
of the initial Gram’s stain, a limited number of
tests were chosen and used to identify each
individual pathogen following rapid culture
(Fig. 1). Certain bacteria, e.g., Streptococcus pyog-
enes, could be identified to the species level, while
others could only be identified to the genus level
(e.g., Enterococcus spp.) or to the family level (e.g.,
certain members of the Enterobacteriaceae).
Antibiotic and diagnostic disks were used to
identify bacteria. In order to confirm the results of
the initial Gram’s stain, aztreonam and linezolid
disks were used. When Strep. pneumoniae was
suspected, an optochin disk was placed on blood
agar plates in later phases of the study, and this
procedure identified correctly all 35 pneumococci
[5]. Catalase and oxidase tests were used for
preliminary identification of Corynebacterium spp.
and Neisseria meningitidis.
Samples from positive anaerobic blood culture
vials were also inoculated on brucella agar plates
with metronidazole and vancomycin disks and
incubated anaerobically for 4 h. An inhibition
zone around the metronidazole disk, in combina-
tion with an absence of aerobic growth, indicated
the presence of anaerobic bacteria. During the
study period, five rapidly growing strains of
Clostridium and Bacteroides spp. were identified.
However, five other, slow-growing anaerobic
isolates did not grow after incubation for 4 h.
The rapid culture and identification method
yielded preliminary identification results for 496
(92%) of the 541 blood cultures within 5 h. Twenty
(4%) isolates did not yield sufficient growth after
4 h, and a further 25 isolates grew, but 15 (3%)
could not be identified using the present algo-
rithm (Fig. 1). Ten (2%) isolates were misdiag-
nosed using the current algorithm (Table 1). The
subsequent use of additional complementary
tests (e.g., optochin disk and L-pyrrolidonyl-
b-naphthylamide tests) prevented misdiagnoses
in the later phases of the study. Identification to
the species level was possible for 192 (39%)
samples, with a further 139 (28%) and 165 (33%)
isolates being identified to the group and family
levels, respectively. The method was also success-
ful with nine samples from sterile body sites other
than blood, comprising four samples from bone
marrow, three cerebrospinal fluids, one peritoneal
dialysis effluent and one synovial fluid.
In conclusion, the present study describes a
rapid 4-h method for preliminary identification of
bacteria in blood culture vials. The method had a
sensitivity of 92%, which made it possible to alert
physicians within 5 h of the automated blood
culture system detecting growth. It was possible
to make a preliminary identification of 67% of the
isolates to the species or group level. The method
was capable of identifying the most frequently
isolated bacteria, i.e., Staph. aureus, coagulase-
negative staphylococci, E. coli, Strep. pneumoniae
and Enterococcus spp., to the species level [6].
Preliminary identification to the group level was
clinically relevant for Enterobactericeae, non-fer-
mentative Gram-negative bacilli and anaerobic
bacteria.
Unfortunately, it was not possible to identify
pathogens such as methicillin-resistant Staph.
aureus, vancomycin-resistant enterococci or
enteric bacteria producing extended-spectrum
b-lactamases. However, these organisms can be
identified using a range of molecular methods.
Another problem concerns possible misidentifica-
tion of certain uncommon pathogens, e.g., Acinet-
obacter, Stenotrophomonas or Listeria. However, the
method described in this report can be established
easily, with <5 min required to perform the spot
tests and a materials cost of only 2–5 Euros per
sample. The method may be used as an alternative
to, or in combination with, molecular techniques
for preliminary rapid species identification of
infecting pathogens in blood cultures.
ACKNOWLEDGEMENTS
M. Kedfors and S. Ahle´n are thanked for excellent technical
assistance. No information has been provided by the authors
concerning the presence or absence of conflicting or dual
interests.
REFERENCES
1. Beekmann SE, Diekema DJ, Chapin KC, Doern GV. Effects
of rapid detection of bloodstream infections on length of
Table 1. Discrepancies in identification between the rapid
culture and conventional identification methods
Rapid culture and identification Conventional method
CoNS Rothia mucilaginosa
CoNS Micrococcus spp.
Group D streptococcus Streptococcus salivarius
Group D streptococcus Enterococcus faecium
Group D streptococcus Enterococcus faecium
Staphylococcus aureus Micrococcus spp.
Enterococcus faecalis Enterococcus faecium
Streptococcus pneumoniae Streptococcus salivarius
Streptococcus pneumoniae a-Haemolytic streptococcus
a-Haemolytic streptococcus Streptococcus pneumoniae
CoNS, coagulase-negative staphylococci.
Research Notes 179
 2007 The Authors
Journal Compilation  2007 European Society of Clinical Microbiology and Infectious Diseases, CMI, 14, 174–189
hospitalization and hospital charges. J Clin Microbiol 2003;
41: 3119–3125.
2. Peters RP, van Agtmael MA, Danner SA, Savelkoul PH,
Vandenbroucke-Grauls CM. New developments in the
diagnosis of bloodstream infections. Lancet Infect Dis 2004; 4:
751–760.
3. de Cueto M, Ceballos E, Martinez-Martinez L, Perea EJ,
Pascual A. Use of positive blood cultures for direct identi-
fication and susceptibility testing with the Vitek 2 system.
J Clin Microbiol 2004; 42: 3734–3738.
4. Kilian M, Bulow P. Identification of Enterobacteriaceae. II.
Use of a beta-glucuronidase detecting agar medium (PGUA
agar) for the identification of E. coli in primary cultures of
urine samples. Acta Pathol Microbiol Scand 1979; 87: 271–276.
5. Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH,
eds. Manual of clinical microbiology, 8th edn. Washington,
DC: ASM Press, 2003.
6. Cockerill FR, Wilson JW, Vetter EA et al. Optimal testing
parameters for blood cultures. Clin Infect Dis 2004; 38: 1724–
1730.
RESEARCH NOTE
Risk-factors for emerging bloodstream
infections caused by extended-spectrum
b-lactamase-producing Escherichia coli
J. Rodrı´guez-Ban˜o1,3, M. D. Navarro1,
L. Romero2, M. A. Muniain1,3, M. de
Cueto2,4, J. Ga´lvez1,3, E. J. Perea2,4 and
A. Pascual2,4
1Seccio´n de Enfermedades Infecciosas, Servicio
de Medicina Interna, 2Servicio de Microbiologı´a,
Hospital Universitario Virgen Macarena,
3Departamento de Medicina and 4Departamento
de Microbiologı´a, Universidad de Sevilla,
Sevilla, Spain
ABSTRACT
Risk-factors for bloodstream infections caused by
extended-spectrum b-lactamase (ESBL)-produc-
ing Escherichia coli were investigated using an
exploratory case-double control study in which 43
cases (70% producing CTX-M enzymes) were
compared with: (i) 86 patients with bacteraemia
caused by non-ESBL-producing E. coli; and (ii) 86
hospitalised patients. Previous follow-up as an
outpatient, urinary catheterisation and use of
oxyimino-b-lactams or fluoroquinolones were
independent risk-factors for ESBL-producing
E. coli among patients with E. coli bacteraemia,
and previous use of oxyimino-b-lactams or fluoro-
quinolones were also independent risk-factors
among hospitalised patients. These findings may
help in identifying patients at greater risk for
bloodstream infection caused by ESBL-producing
E. coli in endemic areas.
Keywords Bacteraemia, Escherichia coli, extended-
spectrum b-lactamases, hospitalised patients, risk-fac-
tors
Original Submission: 7 July 2007; Revised Submis-
sion: 5 August 2007; Accepted: 31 August 2007
Clin Microbiol Infect 2008; 14: 180–183
10.1111/j.1469-0691.2007.01884.x
Escherichia coli producing extended-spectrum
b-lactamases (ESBLs) are an emerging cause of
community and nosocomial infection, and are
associated particularly with the worldwide spread
of CTX-M types of ESBL [1,2]. The treatment
options for patients with infections caused by these
organisms are limited. As is the case with ESBL-
producing Klebsiella pneumoniae [3], treatment with
cephalosporins or fluoroquinolones of blood-
stream infections caused by ESBL-producing
E. coli is associated with a poorer prognosis than
is carbapenem therapy [4]. As bacteraemia caused
by E. coli is frequent, this could result in a signif-
icant increase in the use of carbapenems, which
might contribute to further spread of carbapenem
resistance. Knowledge of risk-factors for bacter-
aemia caused by ESBL-producing E. coli might
help to identify patients at higher risk, who should
therefore receive empirical coverage against these
organisms, and could also permit the identification
of potential intervention strategies. The objective of
this study was therefore to investigate the risk-
factors for bacteraemia caused by ESBL-producing
E. coli in a non-epidemic situation.
A case-double control study with prospective
recruitment was carried out in the Hospital Uni-
versitario Virgen Macarena, a 950-bed hospital in
Sevilla, Spain that provides care for a population of
550 000. The case-patient group included all 43
Corresponding author and reprint requests: J. Rodrı´guez-
Ban˜o, Seccio´n de Enfermedades Infecciosas, Hospital Univer-
sitario Virgen Macarena, Avda Dr Fedriani 3, 41071 Sevilla,
Spain
E-mail: jrb@nacom.es
180 Clinical Microbiology and Infection, Volume 14 Number 2, February 2008
 2007 The Authors
Journal Compilation  2007 European Society of Clinical Microbiology and Infectious Diseases, CMI, 14, 174–189


Rapid culture and identification: a practical method for early preliminary laboratory diagnosis of sepsis 

file:///data/core-remote/dit/data/elsevier/pdf/cab/aHR0cDovL2FwaS5lbHNldmllci5jb20vY29udGVudC9hcnRpY2xlL3BpaS9zMTE5ODc0M3gxNDYzMzQ0OQ==.pdf

Rapid culture and identification: a practical method for early preliminary laboratory diagnosis of sepsis

Abstract

Similar works

Full text

Available Versions

Elsevier - Publisher Connector

Elsevier - Publisher Connector