Journal of Microbiological Methods 109 (2015) 4955

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Journal of Microbiological Methods

journal homepage: www.elsevier.com/locate/jmicmeth

A new approach to determine the susceptibility of bacteria to antibiotics

directly from positive blood culture bottles in two hours
Gabriel A. March a,c,, Mara C. Garca-Loygorri b,1, Mara Simarro a,2, Mara P. Gutirrez a,2,
Antonio Ordua a,c,2,3, Miguel A. Bratos a,c,2,3
a
Department of Microbiology, Faculty of Medicine, University of Valladolid, Av. Ramn y Cajal No. 7, 47005 Valladolid, Spain
b
Service of Microbiology and Parasitology, Medina del Campo Hospital, C/Pearanda No. 4, 47400 Medina del Campo, Spain
c
Service of Microbiology and Immunology, University Clinic Hospital of Valladolid, Ramn y Cajal Avenue No. 3, 47003 Valladolid, Spain

a r t i c l e i n f o a b s t r a c t

Article history: The rapid identication and antibiotic susceptibility test of bacteria causing bloodstream infections are given a
Received 15 October 2014 very high priority by clinical laboratories. In an effort to reduce the time required for performing antibiotic
Received in revised form 6 December 2014 susceptibility test (AST), we have developed a new method to be applied from positive blood culture bottles.
Accepted 8 December 2014
The design of method was performed using blood culture bottles prepared articially with ve strains which
Available online 19 December 2014
have a known susceptibility. An aliquot of the blood culture was subcultured in the presence of specic antibiotics
Keywords:
and bacterial counts were monitored using the Sysmex UF-1000i ow cytometer at different times up to 180 min.
Flow cytometry Receiver operating curve (ROC) analysis allowed us to nd out the cut-off point for differentiating between
Rapid antibiotic susceptibility test sensitive and resistant strains to the tested antibiotic. This procedure was then validated against standard
Blood culture bottles commercial methods on a total of 100 positive blood culture bottles from patients. First, bacterial identication
was performed by matrix-assisted laser desorption ionization-time of ight mass spectrometry (MALDI-TOF-MS)
directly from positive blood culture bottles as we have previously reported. Secondly, antibiotic susceptibility test
was performed in the same way that was carried out in articially prepared blood culture bottles. Our results
indicate that antibiotic susceptibility test can be determined as early as 120 min since a blood culture bottle is
agged as positive. The essential agreement between our susceptibility test and commercial methods (E-test,
MicroScan and Vitek) was 99%. In summary, we conclude that reliable results on bacterial identication and
antibiotic susceptibility test performed directly from positive blood culture bottles can be obtained within 3 h.
2014 Published by Elsevier B.V.

1. Introduction
Sepsis and septic shock are common syndromes among patients
admitted to intensive care units, and are frequently associated with
blood stream infections (Balk, 2000). Several authors have concluded
that early administration of appropriate antibiotics in patients with
sepsis results in increase of survival and reduction of costs of hospitali-
zation (Larche et al., 2003; Kumar et al., 2006; Gaieski et al., 2010;
Barenfanger et al., 1999). In this sense, one of the challenges faced by
clinical laboratories is to reduce the time needed for the identication
as well as antibiotic susceptibility testing of the bacteria responsible
Corresponding author at: Department of Microbiology, Faculty of Medicine, University
of Valladolid, Av. Ramn y Cajal No. 7, 47005 Valladolid, Spain. Tel.: +34 983423023, +34
983420000; fax: +34 983423022, +34 983257511.
E-mail addresses: gmr810@hotmail.com (G.A. March), cgarcialoygorri@saludcastillayleon.es
(M.C. Garca-Loygorri), msimarrogrande@gmail.com (M. Simarro), gutierre@med.uva.es
(M.P. Gutirrez), orduna@med.uva.es (A. Ordua), bratos@med.uva.es (M.A. Bratos).
1
Tel.: +34 983838000; fax: +34 983838007.
2
Tel.: +34 983423023; fax: +34 983423022.
3
Tel.: +34 983420000; fax: +34 983257511.
for a severe infection. In order to obtain results within a short time
frame, several methods have been applied from positive blood culture
bottles, such as mass spectrometry (Lu et al., 2012), ow cytometry
(FCM) (Alvarez-Barrientos et al., 2000), molecular detection techniques
(Rossney et al., 2008), and several commercial systems designed to
perform the identication and susceptibility test (Chen et al., 2008;
Bruins et al., 2004; Funke and Funke-Kissling, 2004; Ling et al., 2003;
Quesada et al., 2010; Lupetti et al., 2010; Gherardi et al., 2012). Thus,
it is possible to perform bacterial identication directly from positive
blood culture within 1 h by using the matrix-assisted laser desorption
ionization-time of ight (MALDI-TOF) system (March-Rossello et al.,
2013; La Scola and Raoult, 2009; Moussaoui et al., 2010; Schubert
et al., 2011). For determination of susceptibility, the molecular detection
techniques can also provide data on only one or few antibiotics in each
determination (Pence et al., 2013); on the other hand, a time period of
at least 11 h is required to obtain the determination of bacteria suscep-
tibility to several antimicrobial agents (Chen et al., 2008; Bruins et al.,
2004; Funke and Funke-Kissling, 2004; Ling et al., 2003; Quesada
et al., 2010; Lupetti et al., 2010; Gherardi et al., 2012). In this context,
the aim of this work was to develop a new approach to perform a faster

http://dx.doi.org/10.1016/j.mimet.2014.12.007
0167-7012/ 2014 Published by Elsevier B.V.
50 G.A. March et al. / Journal of Microbiological Methods 109 (2015) 4955

antibiotic susceptibility test (AST) from positive blood culture bottles by with 1% Pluronic (Sigma-Aldrich, St. Louis, MO, USA). Control tubes
FCM in which the major antibiotics used in sepsis were tested against without antibiotics were included in all experiments.
the most common bacteria isolated from blood. The Sysmex UF-1000i ow cytometer (Sysmex Corporation, Kobe,
Japan) was used to determine bacterial counts in each culture prepared.
2. Material and methods This system is used in clinical laboratories for urine screening. When a
sample is introduced in the system, it is diluted and stained in two
2.1. Ethics statement different reaction chambers, one for bacteria and one for all other
urine particles, which prevents interference with red blood cells and im-
As a result of the consultation performed for the Unidad de proves the detection of bacteria. The staining agent is a uorescent
Investigacin Biomdica of the University Clinical Hospital of Valladolid polymethine dye that binds to deoxyribonucleic acid (DNA). After
(Spain), a waiver was obtained for performing the study protocol of pa- staining, the particles are transported to a ow cell and are irradiated
tients since the nal objective of the study was obtaining non human by a semiconducting laser ( = 635 nm). Forward scatter, side scatter,
material (bacteria). Immediately after blood culture bottles from and uorescence intensities of the individual particles are detected
patients were agged as positive, we anonymized and assigned random and give information about particle size and structure, which is used
numbers to them. Moreover, we have not had access to any identifying to identify and count the particles (Broeren et al., 2011). The linearity
information about patients. We only had access to the information range for microbial count is from 1104 to 1108 colony forming units
about the name and the susceptibility of the bacteria isolated. With (CFU)/ml (van der Zwet et al., 2010).
respect to healthy blood donors, a document including informed In order to calculate the inoculum of the culture control without an-
consent was signed. tibiotic that provided microbial counts at the inoculation time (T0) and
after 3 h of incubation (T3) within the linearity range of the Sysmex
2.2. Experimental design of the antimicrobial susceptibility test from UF-1000i, 50-l aliquots from each articially inoculated blood culture
articially prepared blood culture bottles bottle were added to 30 ml of culture medium, and the microbial con-
centration was determined at T0 and T3. Thus, the initial concentration
In order to set up our method, blood culture bottles were prepared for each bacterial group was established and the same inoculum was
articially with the collection strains Enterococcus faecalis ATCC 29212, also used for each test tube containing the different antibiotic concen-
Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC trations to be tested.
33853, Escherichia coli ATCC 35218 and E. coli ATCC 25922 because Once the T0 bacterial concentration was determined, the tubes con-
these strains show a known susceptibility to antibiotics and, conse- taining the different antibiotic concentrations to be tested together with
quently, they are commonly used as quality control for antibiotic sus- the control tube without antibiotic were incubated in a water bath at
ceptibility testing. To this end, BD BACTECTM Plus Aerobic blood 35 C, and three readings were performed after 1 (T1), 2 (T2) and 3
culture bottles/F Culture Vials (Becton Dickinson, Maryland, USA) (T3) hours of incubation. In order to differentiate between sensitivity
were inoculated with 150 l of a bacterial suspension at 0.5 McFarland and resistance of a strain to the tested antibiotic, bacterial counts
equivalence turbidity of each bacteria strain and with 5 ml of whole obtained for each antibiotic concentration at different times were
blood from healthy volunteer donors. The bottles were incubated into compared, through receiver operating curve (ROC) analysis, with
the Bactec System (Becton Dickinson). The study of antimicrobial sus- those obtained from the control without antibiotic at the same incuba-
ceptibility directly from blood culture bottles prepared articially was tion times. In this way, the optimal threshold for discriminating
started at the time when the incubation system provided a positive between susceptibility and resistance was obtained.
reading and the presence of only a single type of microorganism was Finally, the articially prepared blood culture bottles were harvested
detected by direct microscopic observation. on Columbia agar plates supplemented with 5% sheep blood
The most commonly used antibiotics in clinical practice were tested (bioMrieux, Marcy l'Etoile, France), MacConkey medium (bioMrieux)
at the concentrations of breakpoints described by the Clinical and and agar chocolate PolyVitex (bioMrieux). The plates were incubated
Laboratory Standards Institute (CLSI). Stock solutions of antibiotics at 37 C with 5% CO2 for 24 h. Once the colonies were grown, the
(Vancomycin, Oxacillin, Ceftazidime, Amikacin, Colistin, Ampicillin, bacterial identication of all strains was conrmed by MALDI-TOF
Cefotaxime and Ciprooxacin) were prepared according to the protocol (MALDI Microex LT, Bruker Daltonics, Bremen, Germany) and the
proposed by the CLSI (Clinical and Laboratory Standards Institute, susceptibility was conrmed by means of the commercial methods
2012). The solutions were sterilized by ltration using a MILLEX GS E-test (bioMrieux), VITEK2 (bioMrieux) and MicroScan (Siemens,
0.22 m lter (EMD Millipore Corporation, Billerica, MA, USA) and New York, USA), and minimal inhibitory concentrations (MIC) obtained
stored at 80 C. The antibiotic concentrations tested with the different were interpreted according to criteria published by the CLSI (Clinical
strains are summarized in Table 1. The culture medium used was cation- and Laboratory Standards Institute, 2012).
adjusted MuellerHinton broth (Difco, Sparks, MD, USA) supplemented
2.3. Validation of the antibiotic susceptibility test from positive blood
Table 1 culture bottles of patients
Bacterial strains and the concentrations of the antibiotics used to set up our method.
For validation of the methodology, the sample size was determined
Strain Antibiotic tested Concentrations (mg/L)
based on the population of 1-year positive blood cultures (1256 in
Enterococcus faecalis ATCC 29212 Ampicillin 8, 16 2012; data from the Department of Microbiology at University Clinical
Vancomycin 4, 8, 16, 32
Staphylococcus aureus ATCC 29213 Amikacin 16, 32, 64
Hospital of Valladolid, Spain). By accepting an alpha-risk of 0.95 for a
Cefotaxime 8, 16, 32, 64 precision of 0.1 unit in a bilateral contrast for a proportion estimated
Vancomycin 2, 4, 8, 16 of 0.5 for validation of the susceptibility test applied to blood cultures,
Oxacillin 2, 4 a random sampling of 92 subjects was required. Replacement rate was
Pseudomonas aeruginosa ATCC 27853 Ceftazidime 8, 16, 32
0%. In the present study, a total of 100 positive blood culture bottles of
Amikacin 16, 32, 64
Colistin 2, 4, 8 patients were evaluated for both bacterial identication and susceptibil-
Escherichia coli ATCC 35218 Ampicillin 8, 16, 32 ity testing.
Escherichia coli ATCC 25922 Amikacin 16, 32, 64 Fig. 1 illustrates the work ow diagram executed to perform an AST
Cefotaxime 1, 2, 4 from positive blood culture bottles of patients. When a blood culture
Ciprooxacin 1, 2, 4
bottle was agged as positive, the presence of only one type of
 G.A. March et al. / Journal of Microbiological Methods 109 (2015) 4955
Fig. 1. Work ow diagram of the proposed antibiotic susceptibility test from positive blood
culture bottles of patients.
microorganism was veried by microscopy. Then, direct bacterial
identication was carried out by MALDI-TOF following our previously
described protocol (March-Rossello et al., 2013). After direct identica-
tion was obtained, testing of an antibiotic for each strain present in each
blood culture was performed in the same way as described for the
articially prepared blood cultures. The bacteria identied directly
from a blood culture bottle with the corresponding antibiotic tested
are summarized in Table 2.
Finally, the positive blood culture bottles were harvested as
described for the articially prepared blood cultures. Once the colonies
were grown, the bacterial identication was performed using MALDI-
TOF. This identication was 100% coincident with the direct identica-
tion. Moreover, susceptibility to antibiotics was tested by means of the
commercial methods E-test (bioMrieux), VITEK2 (bioMrieux) and
MicroScan (Siemens, New York, USA), and minimal inhibitory
concentrations (MIC) obtained were interpreted according to criteria
published by the CLSI (Clinical and Laboratory Standards Institute,
2012). The results obtained from these commercial methods were
considered the gold standard of susceptibility. These results were
compared with those obtained by FCM; for this purpose, agreements
and disagreements among the susceptibility values obtained were
classied as agreements, very major errors (false susceptibility), major
errors (false resistance), or minor errors (susceptible/resistant versus
intermediate susceptibility), as recommended by the FDA (Food and
Drug Administration, 2009). Moreover, Kappa concordance index
was calculated to analyze the degree of disagreement with the gold
standard test.
All the statistical calculations were performed using the SPSS v.20.0
software.
51
3. Results
3.1. Results obtained from articially prepared blood culture bottles
In order to obtain all bacterial count readings during the 3 h of
incubation time within the linear range of the Sysmex UF-1000i, we
rst estimated the proper starting concentration (at T0) for each type of
bacteria used. Required starting bacterial concentrations for enterobacteria
and enterococci were in the range of 4104 to 9104 bacteria/ml, and
those for staphylococci and non-fermenting Gram-negative rods
(NFGNR) in the range of 5105 to 9105 bacteria/ml. An inoculum of
50500 l from the positive blood culture bottle was used in all the
cases.
Fig. 2 shows the growth curves of E. faecalis ATCC 29212 when it was
incubated with and without Ampicillin. In the control without antibiot-
ic, it was observed that the microbial count increased nearly one loga-
rithm per hour of incubation. However, when this Ampicillin-sensitive
strain was incubated in the presence of such antibiotic it was observed
that bacterial counts remained lower than those obtained without
antibiotic during the 3 h of incubation. Bacterial counts of E. faecalis
ATCC 29212 after 60 min of incubation in the presence of Ampicillin
were signicantly reduced compared with control (considered as
100%) and, in subsequent readings, after 120 and 180 min of incubation,
this reduction increased gradually (Fig. 3 and Table 3). Similar patterns
were observed in the growth curves of E. faecalis ATCC 29212,
P. aeruginosa ATCC 33853 and E. coli ATCC 25922 strains, which were
sensitive to the antibiotics tested (Table 3). The only resistant strain
tested was E. coli ATCC 35218; this strain was resistant to Ampicillin,
and the growth curves obtained in the presence of antibiotic showed
that counts were similar to those of the control. In the case of S. aureus
ATCC 29213, which was sensitive to the antibiotics tested, we observed
a signicant reduction in bacterial counts compared to control only at
the 2 hour and 3 hour time points (Table 3).
In order to obtain the optimal cut-off bacterial count for discriminat-
ing between sensitivity and resistance, data presented in Table 3 were
analyzed by ROC curve. Table 4 shows a complete sensitivity/specicity
report and indicates the cut-off value in bold. This value allowed us to
consider enterobacteria, enterococci or NFGNR sensitive to the antibiot-
ic tested if a bacterial count reduction of at least 14% compared to
control is observed after 60 min of incubation in the presence of such
antibiotic at a concentration less than or equal to the stated breakpoint
concentration for sensitivity. On the other hand, we considered
enterobacteria, enterococci or NFGNR resistant to the antibiotic tested
if in order to obtain a bacterial count reduction of at least 14% compared
to control after 60 min of incubation, the antibiotic concentration
required is higher than or equal to the stated breakpoint concentration
for resistance. Lastly, enterobacteria, enterococci or NFGNR were
intermediate to the antibiotic tested if neither the sensitivity criterion
nor the resistance criterion were fullled. For staphylococci, the same
interpretation could be applied but using the readings obtained after
120 min of culture incubation.
Applying the aforementioned cut-off, a 100% concordance between
the results of susceptibility obtained by the FCM method and by the
commercial methods (VITEK2, MicroScan and E-test) was observed in
the strains used in the design of the present method (Table 3).
3.2. Results obtained from positive blood culture bottles from patients
In order to asses this method against commercial methods, 100
positive monobacterial blood culture bottles from patients of Valladolid
University Hospital were processed. The results of direct identication
from positive blood culture bottles were 100% coincident with the
results of identication obtained from colonies grown in the subcul-
ture. Besides, the results of the susceptibility tests performed by
commercial methods (VITEK 2, MicroScan and E-test) from colonies
52 G.A. March et al. / Journal of Microbiological Methods 109 (2015) 4955

Table 2
Bacterial strains directly identied from patient blood culture bottles and the concentrations of the antibiotics used to evaluate the accuracy and reliability of our method.

Bacteria Number of strains tested Antibiotic tested Concentrations (mg/L)

Enterococcus faecalis 8 Ampicillin 8, 16

Enterococcus faecium 2
Enterococcus faecalis 4 Vancomycin 4, 8, 16, 32
Staphylococcus hominis 4
Staphylococcus epidermidis 3
Staphylococcus haemolyticus 1
Staphylococcus aureus 1 2, 4, 8, 16
Staphylococcus epidermidis 6 Oxacillin 0.25, 0.5
Staphylococcus hominis 4
Staphylococcus haemolyticus 1
Staphylococcus aureus 2 2, 4
Escherichia coli 4 Ciprooxacin 1, 2, 4
Klebsiella oxytoca 2
Enterobacter kobei 2
Serratia marcescens 2
Klebsiella pneumoniae 1
Enterobacter cloacae 1
Pseudomonas aeruginosa 3 Amikacin 16, 32, 64
Staphylococcus hominis 2
Staphylococcus haemolyticus 1
Staphylococcus aureus 1
Staphylococcus epidermidis 1
Escherichia coli 2
Klebsiella oxytoca 2
Klebsiella pneumoniae 1
Enterobacter kobei 1
Enterobacter cloacae 1
Serratia marcescens 1
Acinetobacter baumannii 1 Cefotaxime 8, 16, 32, 64
Staphylococcus hominis 2
Staphylococcus epidermidis 2
Escherichia coli 3 1, 2, 4
Klebsiella oxytoca 2
Enterobacter kobei 2
Klebsiella pneumoniae 1
Serratia marcescens 1
Enterobacter cloacae 1
Pseudomonas aeruginosa 5 Ceftazidime 8, 16, 32
Pseudomonas stutzeri 1
Pseudomonas putida 1
Acinetobacter baumannii 6 Colistin 2, 4
Acinetobacter johnsonii 1
Acinetobacter ursingii 1
Pseudomonas aeruginosa 5 2, 4, 8
Pseudomonas putida 1

obtained in the subculture of these blood cultures showed 100%

concordance among them.
The susceptibility test performed by FMC from 25 blood culture
bottles containing NFGNR (Supplementary Table 5), 14 blood culture
bottles containing enterococci (Supplementary Table 6) and 31 blood
culture bottles containing staphylococci (Supplementary Table 7)
showed 100% concordance with the susceptibility results obtained by
the commercial methods. The susceptibility test performed from blood
cultures containing enterobacteria showed 96.7% concordance between
the results obtained by FCM and the results obtained by the commercial
methods, since there was only a major error in one of the nine strains
sensitive to Cefotaxime (Supplementary Table 8); this discrepancy
was due to a strain of E. coli, that showed an interpretation as sensitive
by the commercial methods with a MIC of 0.12 mg/L, and an interpreta-
tion as resistant by FCM. Therefore, in the susceptibility test performed
from 100 positive blood culture bottles, there was a 99% concordance
between FCM and the commercial methods, in such a way that the
Kappa index calculation provided a value of 0.9668 (condence interval
95%: 0.90221.0000) (p b 0.001).

4. Discussion
Fig. 2. Bacterial counts of Enterococcus faecalis ATCC 29212 at different incubation times
with Ampicillin at concentrations of breakpoints of sensitivity (8 g/ml) and resistance Several techniques have been applied in order to reduce the time
(16 g/ml) and without Ampicillin. necessary to perform susceptibility testing. MALDI-TOF MS is a technique
 G.A. March et al. / Journal of Microbiological Methods 109 (2015) 4955 53

Table 4
Coordinates of the ROC curve for discriminating between sensitivity and resistance
calculated by SPSS v.20.0 software.

Hypothetical bacterial counts expressed as the Sensitivity Specicity

percentage of the controls without added
antibiotics (100%)

43.1000 1.000 0.000

47.4500 1.000 0.083
54.3500 1.000 0.167
58.2500 1.000 0.250
64.9000 1.000 0.333
71.7000 1.000 0.427
72.3500 1.000 0.500
73.4500 1.000 0.583
74.9000 1.000 0.750
75.6500 1.000 0.833
76.2000 1.000 0.917
86.0500 1.000 1.000
96.6000 0.000 1.000

Fig. 3. Bacterial counts of Enterococcus faecalis ATCC 29212 at different incubation times in
presence of Ampicillin at concentrations of breakpoints of sensitivity (8 g/ml) and resis-
tance (16 g/ml), expressed as percentage of bacterial counts of control without antibiotic
at the same incubation times.
which allows for discrimination between sensitive and resistant strains to
a particular antibiotic (Lu et al., 2012; Kempf et al., 2012; Hrabak et al.,
2011; Sparbier et al., 2012; Camara and Hays, 2007; Edwards-Jones
et al., 2000). However, as these susceptibility studies have been
performed from colonies, there was a delay in the diagnosis of 17 h
compared to the susceptibility test performed in our study. Furthermore,
the routine use of MALDI-TOF system is not feasible to perform an AST
because it cannot be used for all groups of antibiotics, there is still no
uniform methodology and it is necessary to create one's own databases.
An inconvenience of the molecular techniques for detection of antimicro-
bial resistance is that they are analytical methods designed to identify a
single antibiotic resistant gene (Pence et al., 2013). On the other hand,
for performing an antibiotic susceptibility test from positive blood
cultures, various commercial methods, which are routinely applied for
susceptibility determination from colonies, were also used. Thus, 11 to
35 h was needed to obtain the susceptibility results under a consistency
from 77% to practically 100% (Chen et al., 2008; Bruins et al., 2004;
Funke and Funke-Kissling, 2004; Ling et al., 2003; Quesada et al., 2010;
Lupetti et al., 2010; Gherardi et al., 2012); this time period was much
longer than that required for our study.
FCM is a useful technique to test susceptibility of bacteria to antimi-
crobial agents. These studies are based on the effects of antimicrobial
agents on certain metabolic parameters of microorganisms, i.e.: mem-
brane integrity (Ramani and Chaturvedi, 2000), light scattering
(Shrestha et al., 2011) or enzymatic activity (Kirk et al., 1998). These
studies were performed from colonies grown in isolation plates and
none of such methods have succeeded due to their complicated proce-
dure and high cost (Broeren et al., 2013). On the other hand, few studies
have been published from direct samples (exudates, urines) (Gauthier
et al., 2002; Cohen and Sahar, 1989), but none of them have been
performed directly from positive blood culture bottles.
Here we present a new approach to determine the susceptibility of
bacteria to antibiotics directly from positive blood culture bottles by
FCM. The rst step of the proposed AST consisted of identifying the
bacteria being tested because interpretation of MIC depends on the
bacterial species. For this reason, direct bacterial identication from
positive blood culture of patients was performed by MALDI-TOF
following our previously published protocol (March-Rossello et al.,
2013), which allows obtaining direct identication in 1 h. If direct

Table 3
Percentage of bacterial counts obtained from collection strains incubated in the presence of the indicated concentrations of antibiotics regarding bacterial counts of the controls without
antibiotics (100%) after different incubation times.

Strain tested Antibiotic Susceptibility by Incubation times Susceptibility from blood

tested commercial culture
0 min 60 min 120 min 180 min
methodsa bottle by ow cytometry
Sb I R S I R S I R S I R

E. faecalis 29212 Ampicillin Sensitive 101.2 97.8 66.1 57.3 10.3 9.6 4.8 5.6 Sensitive
Vancomycin Sensitive 100.5 99.4, 102.5 71.2 72.5, 65.9 54.6 51.0, 43.4 11.5 9.7, 8.6 7.2 Sensitive
98.7c 70.5 47.6
S. aureus 29213 Amikacin Sensitive 99.7 102.3 101.2 91.6 92.1 92.8 44.4 41.7 48.5 10.7 8.6 10.0 Sensitive
Cefotaxime Sensitive 98.5 99.4, 99.1 94.4 96.0, 95.9 57.9 60.2, 59.5 25.1 24.3, 30.0 Sensitive
98.8 96.2 63.2 23.9
Vancomycin Sensitive 102.2 98.5, 101.6 95.2 96.1, 96.9 76.5 72.6, 70.6 18.2 20.0, 19.9 Sensitive
99.9 97.9 71.1 21.2
Oxacillin Sensitive 100.4 99.1 96.6 96.9 74.4 73.3 27.4 - 28.1 Sensitive
P. aeruginosa Ceftazidime Sensitive 98.7 103.6 100.4 75.9 76.2 73.5 33.7 32.0 31.7 13.6 13.3 10.2 Sensitive
27853 Amikacin Sensitive 103.4 100.9 104.1 72.5 68.5 69.6 25.3 24.2 21.5 7.2 5.3 4.8 Sensitive
Colistin Sensitive 98.4 102.7 97.7 74.4 75.2 73.8 44.4 43.9 43.1 19.4 18.8 17.5 Sensitive
E. coli 35218 Ampicillin Resistant 103.2 98.4 99.9 95.6 97.3 96.9 92.7 94.0 93.2 89.9 88.3 86.5 Resistant
E. coli 25922 Amikacin Sensitive 97.3 98.0 99.7 72.2 73.6 73.9 15.5 14.9 14.0 0.1 0.2 2.1 Sensitive
Cefotaxime Sensitive 102.8 99.1 98.4 50.8 52.0 49.3 5.6 4.3 3.8 0.1 0.1 0.1 Sensitive
Ciprooxacin Sensitive 98.3 99.4 102.7 75.4 74.9 74.2 27.6 25.5 24.9 9.7 10.5 7.9 Sensitive
a
Determined concurrently by E-test, VITEK2 and Microscan from colonies grown in culture plates.
b
S, I and R: concentrations of antibiotic at breakpoint of sensitivity, intermediate and resistance tested.
c
Results with the lowest and highest breakpoint concentration of intermediate.
54 G.A. March et al. / Journal of Microbiological Methods 109 (2015) 4955
identication is not achieved, the AST directly from positive blood
culture cannot be performed. In this work, direct bacterial identication
was achieved in the 100 positive blood culture bottles processed, which
corroborated our previous results (March-Rossello et al., 2013). The
novelty of the work lies in that we have developed an AST directly
from blood culture bottles by FCM and that the results can be obtained
sooner than those obtained by other methods previously published.
The Sysmex UF-1000i ow cytometry is a system used in clinical
laboratories for urine screening, and is able to quantify bacteria
(Manoni et al., 2009). This ability is applied in the present study in
order to perform a rapid susceptibility test from a positive blood culture.
In order to reduce the number of bacterial aggregates and obtain better
bacterial counts, tensioactive Pluronic has been added to the cultures.
Thus, it was observed that counts for both Gram-positive and Gram-
negative bacteria were higher with Pluronic than without it (data not
shown).
The interpretation of the susceptibility test was carried out by
comparing the bacterial counts between cultures incubated with and
without antibiotics. Thus, the proposed methodology is based on the
investigation of the phenotypic behavior of bacteria in the presence of
an antibiotic. The phenotypic traits are much more determinant than
the genotypic traits, since, ultimately, the phenotype is the expression
of the bacteria behavior in the patient when antibiotics are administrat-
ed. That is to say, in order to detect the effect that an antibiotic exerts on
bacteria, a lapse of time is necessary to observe if the bacteria can or
cannot grow, depending on their susceptibility to the antimicrobial
agent. Thus, in order to study the susceptibility, the bacterial counts ob-
tained from the strains incubated in the presence of the antibiotic were
compared, by using ROC curves, to the bacterial counts from controls
without antibiotic, at the same times. And it was observed that the
count obtained from enterobacteria, enterococci and NFGNR after 1 h
of incubation made it possible to determine if a bacterium was sensitive
or resistant to the antibiotic. On the other hand, staphylococci needed
2 h of incubation for this determination.
Broeren et al. (2013), by using the Sysmex UF-1000i, performed a
study to obtain the MIC from plate-grown colonies. These authors
dened the MIC by FCM as the lowest concentration of antibiotic that
provided an 80% decrease in the number of bacteria compared with
control without antibiotic after 240 min of incubation. Moreover, they
suggested that a valid result could be obtained in 90 min for E. coli and
in 120 min for P. aeruginosa and S. aureus. In our study, we needed
only 1 h to obtain the susceptibility results for enterococci, NFGNR and
enterobacteria and 2 h in staphylococci. These incubation times are
very similar to those obtained by these authors (Broeren et al., 2013),
but we gained the necessary time to obtain colonies, which is to say,
an average of 17 h.
We found only one major error with regard to the gold standard. The
major error was due to a strain of E. coli tested with Cefotaxime. This test
was repeated twice and the same result was obtained. Despite this
discrepancy, we obtained a very good Kappa index with a value of
0.9668 (condence interval 95%: 0.90221.0000) (p b 0.001). Further-
more, we have not obtained any very major error, which are the errors
that would involve the most severe consequences for the patient, since
an antibiotic would be reported as sensitive when, in fact, the bacteria
are resistant to this antibiotic.
The proposed method allows obtaining a rapid antibiotic susceptibil-
ity test. Nevertheless, we are aware of the limitations of the study; in the
validation procedure, more strains and more antibiotics not included in
this study should be tested. On the other hand, the proposed method
could partially solve the problem derived from the time needed to
report a result in clinical microbiology laboratories given that we have
demonstrated that it is possible to obtain the results on identication
and susceptibility in 2 h for Gram-negative bacteria and enterococci,
and in 3 h for staphylococci. To the best of our knowledge, this is the
shortest time found in the literature needed to perform identication
and susceptibility testing from positive blood cultures. Another
advantage of the proposed susceptibility test is that the antibiotics to
be tested can be selected in accordance with the patient's medical histo-
ry and with the microorganism that causes the infection; furthermore,
this method could also be applied to other monomicrobial samples,
for example urine and others biological uids from normally sterile
sites, and colonies grown in isolation plates. In conclusion, by applying
our AST, patients who suffer blood stream infections could receive the
correct antibiotic within 3 h from the incubation system of blood
culture bottles agged as positive.
5. Conicts of interest
The authors declare that they have no conicts of interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.mimet.2014.12.007.
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