A new approach to determine the susceptibility of bacteria to antibiotics directly from positive blood culture bottles in two hours

Journal of Microbiological Methods 109 (2015) 49–55

Contents lists available at ScienceDirect

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,⁎, María C. García-Loygorri b,1, María Simarro a,2, María P. Gutiérrez a,2, Antonio Orduña a,c,2,3, Miguel A. Bratos a,c,2,3 a b c

Department of Microbiology, Faculty of Medicine, University of Valladolid, Av. Ramón y Cajal No. 7, 47005 Valladolid, Spain Service of Microbiology and Parasitology, Medina del Campo Hospital, C/Peñaranda No. 4, 47400 Medina del Campo, Spain Service of Microbiology and Immunology, University Clinic Hospital of Valladolid, Ramón y Cajal Avenue No. 3, 47003 Valladolid, Spain

a r t i c l e

i n f o

Article history: Received 15 October 2014 Received in revised form 6 December 2014 Accepted 8 December 2014 Available online 19 December 2014 Keywords: Flow cytometry Rapid antibiotic susceptibility test Blood culture bottles

a b s t r a c t The rapid identification and antibiotic susceptibility test of bacteria causing bloodstream infections are given a very high priority by clinical laboratories. In an effort to reduce the time required for performing antibiotic susceptibility test (AST), we have developed a new method to be applied from positive blood culture bottles. The design of method was performed using blood culture bottles prepared artificially with five strains which have a known susceptibility. An aliquot of the blood culture was subcultured in the presence of specific antibiotics and bacterial counts were monitored using the Sysmex UF-1000i flow cytometer at different times up to 180 min. Receiver operating curve (ROC) analysis allowed us to find out the cut-off point for differentiating between sensitive and resistant strains to the tested antibiotic. This procedure was then validated against standard commercial methods on a total of 100 positive blood culture bottles from patients. First, bacterial identification was performed by matrix-assisted laser desorption ionization-time of flight 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 artificially 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 flagged 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 identification 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 hospitalization (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 identification as well as antibiotic susceptibility testing of the bacteria responsible ⁎ Corresponding author at: Department of Microbiology, Faculty of Medicine, University of Valladolid, Av. Ramón y Cajal No. 7, 47005 Valladolid, Spain. Tel.: +34 983423023, +34 983420000; fax: +34 983423022, +34 983257511. E-mail addresses: [email protected] (G.A. March), [email protected] (M.C. García-Loygorri), [email protected] (M. Simarro), [email protected] (M.P. Gutiérrez), [email protected] (A. Orduña), [email protected] (M.A. Bratos). 1 Tel.: +34 983838000; fax: +34 983838007. 2 Tel.: +34 983423023; fax: +34 983423022. 3 Tel.: +34 983420000; fax: +34 983257511.

http://dx.doi.org/10.1016/j.mimet.2014.12.007 0167-7012/© 2014 Published by Elsevier B.V.

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), flow cytometry (FCM) (Alvarez-Barrientos et al., 2000), molecular detection techniques (Rossney et al., 2008), and several commercial systems designed to perform the identification 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 identification directly from positive blood culture within 1 h by using the matrix-assisted laser desorption ionization-time of flight (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 susceptibility 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

50

G.A. March et al. / Journal of Microbiological Methods 109 (2015) 49–55

antibiotic susceptibility test (AST) from positive blood culture bottles by FCM in which the major antibiotics used in sepsis were tested against the most common bacteria isolated from blood. 2. Material and methods 2.1. Ethics statement As a result of the consultation performed for the Unidad de Investigación Biomédica of the University Clinical Hospital of Valladolid (Spain), a waiver was obtained for performing the study protocol of patients since the final objective of the study was obtaining non human material (bacteria). Immediately after blood culture bottles from patients were flagged as positive, we anonymized and assigned random numbers to them. Moreover, we have not had access to any identifying information about patients. We only had access to the information about the name and the susceptibility of the bacteria isolated. With respect to healthy blood donors, a document including informed consent was signed. 2.2. Experimental design of the antimicrobial susceptibility test from artificially prepared blood culture bottles In order to set up our method, blood culture bottles were prepared artificially with the collection strains Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 33853, Escherichia coli ATCC 35218 and E. coli ATCC 25922 because these strains show a known susceptibility to antibiotics and, consequently, they are commonly used as quality control for antibiotic susceptibility testing. To this end, BD BACTECTM Plus Aerobic blood culture bottles/F Culture Vials (Becton Dickinson, Maryland, USA) were inoculated with 150 μl of a bacterial suspension at 0.5 McFarland equivalence turbidity of each bacteria strain and with 5 ml of whole blood from healthy volunteer donors. The bottles were incubated into the Bactec System (Becton Dickinson). The study of antimicrobial susceptibility directly from blood culture bottles prepared artificially was started at the time when the incubation system provided a positive reading and the presence of only a single type of microorganism was detected by direct microscopic observation. The most commonly used antibiotics in clinical practice were tested at the concentrations of breakpoints described by the Clinical and Laboratory Standards Institute (CLSI). Stock solutions of antibiotics (Vancomycin, Oxacillin, Ceftazidime, Amikacin, Colistin, Ampicillin, Cefotaxime and Ciprofloxacin) were prepared according to the protocol proposed by the CLSI (Clinical and Laboratory Standards Institute, 2012). The solutions were sterilized by filtration using a MILLEX GS 0.22 μm filter (EMD® Millipore Corporation, Billerica, MA, USA) and stored at −80 °C. The antibiotic concentrations tested with the different strains are summarized in Table 1. The culture medium used was cationadjusted Mueller–Hinton broth (Difco, Sparks, MD, USA) supplemented Table 1 Bacterial strains and the concentrations of the antibiotics used to set up our method. Strain

Antibiotic tested

Concentrations (mg/L)

Enterococcus faecalis ATCC 29212

Ampicillin Vancomycin Amikacin Cefotaxime Vancomycin Oxacillin Ceftazidime Amikacin Colistin Ampicillin Amikacin Cefotaxime Ciprofloxacin

8, 16 4, 8, 16, 32 16, 32, 64 8, 16, 32, 64 2, 4, 8, 16 2, 4 8, 16, 32 16, 32, 64 2, 4, 8 8, 16, 32 16, 32, 64 1, 2, 4 1, 2, 4

Staphylococcus aureus ATCC 29213

Pseudomonas aeruginosa ATCC 27853

Escherichia coli ATCC 35218 Escherichia coli ATCC 25922

with 1% Pluronic (Sigma-Aldrich, St. Louis, MO, USA). Control tubes without antibiotics were included in all experiments. The Sysmex UF-1000i flow cytometer (Sysmex Corporation, Kobe, Japan) was used to determine bacterial counts in each culture prepared. 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 different reaction chambers, one for bacteria and one for all other urine particles, which prevents interference with red blood cells and improves the detection of bacteria. The staining agent is a fluorescent polymethine dye that binds to deoxyribonucleic acid (DNA). After staining, the particles are transported to a flow cell and are irradiated by a semiconducting laser (λ = 635 nm). Forward scatter, side scatter, and fluorescence intensities of the individual particles are detected and give information about particle size and structure, which is used to identify and count the particles (Broeren et al., 2011). The linearity range for microbial count is from 1·104 to 1·108 colony forming units (CFU)/ml (van der Zwet et al., 2010). In order to calculate the inoculum of the culture control without antibiotic that provided microbial counts – at the inoculation time (T0) and after 3 h of incubation (T3) – within the linearity range of the Sysmex UF-1000i, 50-μl aliquots from each artificially inoculated blood culture bottle were added to 30 ml of culture medium, and the microbial concentration was determined at T0 and T3. Thus, the initial concentration for each bacterial group was established and the same inoculum was also used for each test tube containing the different antibiotic concentrations to be tested. Once the T0 bacterial concentration was determined, the tubes containing the different antibiotic concentrations to be tested together with the control tube without antibiotic were incubated in a water bath at 35 °C, and three readings were performed after 1 (T1), 2 (T2) and 3 (T3) hours of incubation. In order to differentiate between sensitivity and resistance of a strain to the tested antibiotic, bacterial counts obtained for each antibiotic concentration at different times were compared, through receiver operating curve (ROC) analysis, with those obtained from the control without antibiotic at the same incubation times. In this way, the optimal threshold for discriminating between susceptibility and resistance was obtained. Finally, the artificially prepared blood culture bottles were harvested on Columbia agar plates supplemented with 5% sheep blood (bioMérieux, Marcy l'Etoile, France), MacConkey medium (bioMérieux) and agar chocolate PolyVitex (bioMérieux). The plates were incubated at 37 °C with 5% CO2 for 24 h. Once the colonies were grown, the bacterial identification of all strains was confirmed by MALDI-TOF (MALDI Microflex LT, Bruker Daltonics, Bremen, Germany) and the susceptibility was confirmed by means of the commercial methods E-test (bioMérieux), VITEK2 (bioMérieux) 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). 2.3. Validation of the antibiotic susceptibility test from positive blood culture bottles of patients For validation of the methodology, the sample size was determined based on the population of 1-year positive blood cultures (1256 in 2012; data from the Department of Microbiology at University Clinical Hospital of Valladolid, Spain). By accepting an alpha-risk of 0.95 for a precision of 0.1 unit in a bilateral contrast for a proportion estimated of 0.5 for validation of the susceptibility test applied to blood cultures, a random sampling of 92 subjects was required. Replacement rate was 0%. In the present study, a total of 100 positive blood culture bottles of patients were evaluated for both bacterial identification and susceptibility testing. Fig. 1 illustrates the work flow diagram executed to perform an AST from positive blood culture bottles of patients. When a blood culture bottle was flagged as positive, the presence of only one type of

G.A. March et al. / Journal of Microbiological Methods 109 (2015) 49–55

51

3. Results 3.1. Results obtained from artificially prepared blood culture bottles

Fig. 1. Work flow diagram of the proposed antibiotic susceptibility test from positive blood culture bottles of patients.

microorganism was verified by microscopy. Then, direct bacterial identification was carried out by MALDI-TOF following our previously described protocol (March-Rossello et al., 2013). After direct identification was obtained, testing of an antibiotic for each strain present in each blood culture was performed in the same way as described for the artificially prepared blood cultures. The bacteria identified 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 artificially prepared blood cultures. Once the colonies were grown, the bacterial identification was performed using MALDITOF. This identification was 100% coincident with the direct identification. Moreover, susceptibility to antibiotics was tested by means of the commercial methods E-test (bioMérieux), VITEK2 (bioMérieux) 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 classified 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.

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 first 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 4·104 to 9·104 bacteria/ml, and those for staphylococci and non-fermenting Gram-negative rods (NFGNR) in the range of 5·105 to 9·105 bacteria/ml. An inoculum of 50–500 μl 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 antibiotic, it was observed that the microbial count increased nearly one logarithm 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 significantly 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 significant 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 discriminating between sensitivity and resistance, data presented in Table 3 were analyzed by ROC curve. Table 4 shows a complete sensitivity/specificity report and indicates the cut-off value in bold. This value allowed us to consider enterobacteria, enterococci or NFGNR sensitive to the antibiotic 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 fulfilled. 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 identification from positive blood culture bottles were 100% coincident with the results of identification obtained from colonies grown in the subculture. 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) 49–55

Table 2 Bacterial strains directly identified 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 Enterococcus faecium Enterococcus faecalis Staphylococcus hominis Staphylococcus epidermidis Staphylococcus haemolyticus Staphylococcus aureus Staphylococcus epidermidis Staphylococcus hominis Staphylococcus haemolyticus Staphylococcus aureus Escherichia coli Klebsiella oxytoca Enterobacter kobei Serratia marcescens Klebsiella pneumoniae Enterobacter cloacae Pseudomonas aeruginosa Staphylococcus hominis Staphylococcus haemolyticus Staphylococcus aureus Staphylococcus epidermidis Escherichia coli Klebsiella oxytoca Klebsiella pneumoniae Enterobacter kobei Enterobacter cloacae Serratia marcescens Acinetobacter baumannii Staphylococcus hominis Staphylococcus epidermidis Escherichia coli Klebsiella oxytoca Enterobacter kobei Klebsiella pneumoniae Serratia marcescens Enterobacter cloacae Pseudomonas aeruginosa Pseudomonas stutzeri Pseudomonas putida Acinetobacter baumannii Acinetobacter johnsonii Acinetobacter ursingii Pseudomonas aeruginosa Pseudomonas putida

8 2 4 4 3 1 1 6 4 1 2 4 2 2 2 1 1 3 2 1 1 1 2 2 1 1 1 1 1 2 2 3 2 2 1 1 1 5 1 1 6 1 1 5 1

Ampicillin

8, 16

Vancomycin

4, 8, 16, 32

Oxacillin

2, 4, 8, 16 0.25, 0.5

Ciprofloxacin

2, 4 1, 2, 4

Amikacin

16, 32, 64

Cefotaxime

8, 16, 32, 64

1, 2, 4

Ceftazidime

8, 16, 32

Colistin

2, 4

2, 4, 8

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 interpretation 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 (confidence interval 95%: 0.9022–1.0000) (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 μg/ml) and resistance (16 μg/ml) and without Ampicillin.

Several techniques have been applied in order to reduce the time necessary to perform susceptibility testing. MALDI-TOF MS is a technique

G.A. March et al. / Journal of Microbiological Methods 109 (2015) 49–55

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 percentage of the controls without added antibiotics (100%)

Sensitivity

Specificity

43.1000 47.4500 54.3500 58.2500 64.9000 71.7000 72.3500 73.4500 74.9000 75.6500 76.2000 86.0500 96.6000

1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.000

0.000 0.083 0.167 0.250 0.333 0.427 0.500 0.583 0.750 0.833 0.917 1.000 1.000

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 antimicrobial agents. These studies are based on the effects of antimicrobial agents on certain metabolic parameters of microorganisms, i.e.: membrane 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 procedure 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 first 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 identification 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 identification in 1 h. If direct

Fig. 3. Bacterial counts of Enterococcus faecalis ATCC 29212 at different incubation times in presence of Ampicillin at concentrations of breakpoints of sensitivity (8 μg/ml) and resistance (16 μg/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 antimicrobial 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

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 tested

Susceptibility by commercial methodsa

Incubation times 0 min Sb

E. faecalis 29212 Ampicillin Vancomycin

Sensitive Sensitive

S. aureus 29213

Amikacin Cefotaxime

Sensitive Sensitive

Vancomycin

Sensitive

Oxacillin Ceftazidime Amikacin Colistin Ampicillin Amikacin Cefotaxime Ciprofloxacin

Sensitive Sensitive Sensitive Sensitive Resistant Sensitive Sensitive Sensitive

P. aeruginosa 27853 E. coli 35218 E. coli 25922

a b c

60 min I

101.2 – 100.5 99.4, 98.7c 99.7 102.3 98.5 99.4, 98.8 102.2 98.5, 99.9 100.4 – 98.7 103.6 103.4 100.9 98.4 102.7 103.2 98.4 97.3 98.0 102.8 99.1 98.3 99.4

R

S

I

97.8 66.1 – 102.5 71.2 72.5, 70.5 101.2 91.6 92.1 99.1 94.4 96.0, 96.2 101.6 95.2 96.1, 97.9 99.1 96.6 – 100.4 75.9 76.2 104.1 72.5 68.5 97.7 74.4 75.2 99.9 95.6 97.3 99.7 72.2 73.6 98.4 50.8 52.0 102.7 75.4 74.9

120 min R

S

I

57.3 10.3 – 65.9 54.6 51.0, 47.6 92.8 44.4 41.7 95.9 57.9 60.2, 63.2 96.9 76.5 72.6, 71.1 96.9 74.4 – 73.5 33.7 32.0 69.6 25.3 24.2 73.8 44.4 43.9 96.9 92.7 94.0 73.9 15.5 14.9 49.3 5.6 4.3 74.2 27.6 25.5

Determined concurrently by E-test, VITEK2 and Microscan from colonies grown in culture plates. S, I and R: concentrations of antibiotic at breakpoint of sensitivity, intermediate and resistance tested. Results with the lowest and highest breakpoint concentration of intermediate.

Susceptibility from blood culture bottle by flow cytometry

180 min R

S

I

9.6 4.8 – 43.4 11.5 9.7, 8.6 48.5 10.7 8.6 59.5 25.1 24.3, 23.9 70.6 18.2 20.0, 21.2 73.3 27.4 31.7 13.6 13.3 21.5 7.2 5.3 43.1 19.4 18.8 93.2 89.9 88.3 14.0 0.1 0.2 3.8 0.1 0.1 24.9 9.7 10.5

R 5.6 Sensitive 7.2 Sensitive 10.0 Sensitive 30.0 Sensitive 19.9 Sensitive 28.1 10.2 4.8 17.5 86.5 2.1 0.1 7.9

Sensitive Sensitive Sensitive Sensitive Resistant Sensitive Sensitive Sensitive

54

G.A. March et al. / Journal of Microbiological Methods 109 (2015) 49–55

identification is not achieved, the AST directly from positive blood culture cannot be performed. In this work, direct bacterial identification 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 flow 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 Gramnegative 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 administrated. 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 obtained 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 defined 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 (confidence interval 95%: 0.9022–1.0000) (p b 0.001). Furthermore, 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 susceptibility 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 identification 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 identification 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 history 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 fluids 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 flagged as positive. 5. Conflicts of interest The authors declare that they have no conflicts 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. References Alvarez-Barrientos, A., Arroyo, J., Canton, R., Nombela, C., Sanchez-Perez, M., 2000. Applications of flow cytometry to clinical microbiology. Clin. Microbiol. Rev. 13, 167–195. Balk, R.A., 2000. Severe sepsis and septic shock. Definitions, epidemiology, and clinical manifestations. Crit. Care Clin. 16, 179–192. Barenfanger, J., Drake, C., Kacich, G., 1999. Clinical and financial benefits of rapid bacterial identification and antimicrobial susceptibility testing. J. Clin. Microbiol. 37, 1415–1418. Broeren, M.A., Bahceci, S., Vader, H.L., Arents, N.L., 2011. Screening for urinary tract infection with the Sysmex UF-1000i urine flow cytometer. J. Clin. Microbiol. 49, 1025–1029. Broeren, M.A., Maas, Y., Retera, E., Arents, N.L., 2013. Antimicrobial susceptibility testing in 90 min by bacterial cell count monitoring. Clin. Microbiol. Infect. 19, 286–291. Bruins, M.J., Bloembergen, P., Ruijs, G.J., Wolfhagen, M.J., 2004. Identification and susceptibility testing of Enterobacteriaceae and Pseudomonas aeruginosa by direct inoculation from positive BACTEC blood culture bottles into Vitek 2. J. Clin. Microbiol. 42, 7–11. Camara, J.E., Hays, F.A., 2007. Discrimination between wild-type and ampicillin-resistant Escherichia coli by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Bioanal. Chem. 389, 1633–1638. Chen, J.R., Lee, S.Y., Yang, B.H., Lu, J.J., 2008. Rapid identification and susceptibility testing using the VITEK 2 system using culture fluids from positive BacT/ALERT blood cultures. J. Microbiol. Immunol. Infect. 41, 259–264. Clinical and Laboratory Standards Institute (CLSI), 2012. Performance standards for antimicrobial susceptibility testing; twenty-second information supplement vol. 32. no. 3, p. M100-S22. Cohen, C.Y., Sahar, E., 1989. Rapid flow cytometric bacterial detection and determination of susceptibility to amikacin in body fluids and exudates. J. Clin. Microbiol. 27, 1250–1256. Edwards-Jones, V., Claydon, M.A., Evason, D.J., Walker, J., Fox, A.J., Gordon, D.B., 2000. Rapid discrimination between methicillin-sensitive and methicillin-resistant Staphylococcus aureus by intact cell mass spectrometry. J. Med. Microbiol. 49, 295–300. Food and Drug Administration, 2009. Guidance on review criteria for assessment of antimicrobial susceptibility devices. (Available at), http://www.fda.gov/ohrms/ dockets/98fr/000109gd.pdf (Accessed 20 January 2013). Funke, G., Funke-Kissling, P., 2004. Use of the BD PHOENIX Automated Microbiology System for direct identification and susceptibility testing of Gram-negative rods from positive blood cultures in a three-phase trial. J. Clin. Microbiol. 42, 1466–1470. Gaieski, D.F., Mikkelsen, M.E., Band, R.A., Pines, J.M., Massone, R., Furia, F.F., Shofer, F.S., Goyal, M., 2010. Impact of time to antibiotics on survival in patients with severe sepsis or septic shock in whom early goal-directed therapy was initiated in the emergency department. Crit. Care Med. 38, 1045–1053. Gauthier, C., St-Pierre, Y., Villemur, R., 2002. Rapid antimicrobial susceptibility testing of urinary tract isolates and samples by flow cytometry. J. Med. Microbiol. 51, 192–200. Gherardi, G., Angeletti, S., Panitti, M., Pompilio, A., Di Bonaventura, G., Crea, F., Avola, A., Fico, L., Palazzo, C., Sapia, G.F., Visaggio, D., Dicuonzo, G., 2012. Comparative evaluation of the Vitek-2 Compact and Phoenix systems for rapid identification and antibiotic susceptibility testing directly from blood cultures of Gram-negative and Gram-positive isolates. Diagn. Microbiol. Infect. Dis. 72, 20–31. Hrabak, J., Walkova, R., Studentova, V., Chudackova, E., Bergerova, T., 2011. Carbapenemase activity detection by matrix-assisted laser desorption ionizationtime of flight mass spectrometry. J. Clin. Microbiol. 49, 3222–3227. Kempf, M., Bakour, S., Flaudrops, C., Berrazeg, M., Brunel, J.M., Drissi, M., Mesli, E., Touati, A., Rolain, J.M., 2012. Rapid detection of carbapenem resistance in Acinetobacter baumannii using matrix-assisted laser desorption ionization-time of flight mass spectrometry. PLoS One 7, e31676. Kirk, S.M., Schell, R.F., Moore, A.V., Callister, S.M., Mazurek, G.H., 1998. Flow cytometric testing of susceptibilities of Mycobacterium tuberculosis isolates to ethambutol, isoniazid, and rifampin in 24 hours. J. Clin. Microbiol. 36, 1568–1573.

G.A. March et al. / Journal of Microbiological Methods 109 (2015) 49–55 Kumar, A., Roberts, D., Wood, K.E., Light, B., Parrillo, J.E., Sharma, S., Suppes, R., Feinstein, D., Zanotti, S., Taiberg, L., Gurka, D., Cheang, M., 2006. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit. Care Med. 34, 1589–1596. La Scola, B., Raoult, D., 2009. Direct identification of bacteria in positive blood culture bottles by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. PLoS One 4, e8041. Larche, J., Azoulay, E., Fieux, F., Mesnard, L., Moreau, D., Thiery, G., Darmon, M., Le Gall, J.R., Schlemmer, B., 2003. Improved survival of critically ill cancer patients with septic shock. Intensive Care Med. 29, 1688–1695. Ling, T.K., Liu, Z.K., Cheng, A.F., 2003. Evaluation of the VITEK 2 system for rapid direct identification and susceptibility testing of Gram-negative bacilli from positive blood cultures. J. Clin. Microbiol. 41, 4705–4707. Lu, J.J., Tsai, F.J., Ho, C.M., Liu, Y.C., Chen, C.J., 2012. Peptide biomarker discovery for identification of methicillin-resistant and vancomycin-intermediate Staphylococcus aureus strains by MALDI-TOF. Anal. Chem. 84, 5685–5692. Lupetti, A., Barnini, S., Castagna, B., Capria, A.L., Nibbering, P.H., 2010. Rapid identification and antimicrobial susceptibility profiling of Gram-positive cocci in blood cultures with the Vitek 2 system. Eur. J. Clin. Microbiol. Infect. Dis. 29, 89–95. Manoni, F., Fornasiero, L., Ercolin, M., Tinello, A., Ferrian, M., Hoffer, P., Valverde, S., Gessoni, G., 2009. Cutoff values for bacteria and leukocytes for urine flow cytometer Sysmex UF-1000i in urinary tract infections. Diagn. Microbiol. Infect. Dis. 65, 103–107. March-Rossello, G.A., Munoz-Moreno, M.F., Garcia-Loygorri-Jordan de Urries, M.C., Bratos-Perez, M.A., 2013. A differential centrifugation protocol and validation criterion for enhancing mass spectrometry (MALDI-TOF) results in microbial identification using blood culture growth bottles. Eur. J. Clin. Microbiol. Infect. Dis. 32, 699–704. Moussaoui, W., Jaulhac, B., Hoffmann, A.M., Ludes, B., Kostrzewa, M., Riegel, P., Prevost, G., 2010. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identifies 90% of bacteria directly from blood culture vials. Clin. Microbiol. Infect. 16, 1631–1638.

55

Pence, M.A., McElvania TeKippe, E., Burnham, C.A., 2013. Diagnostic assays for identification of microorganisms and antimicrobial resistance determinants directly from positive blood culture broth. Clin. Lab. Med. 33, 651–684. Quesada, M.D., Gimenez, M., Molinos, S., Fernandez, G., Sanchez, M.D., Rivelo, R., Ramirez, A., Banque, G., Ausina, V., 2010. Performance of VITEK-2 Compact and overnight MicroScan panels for direct identification and susceptibility testing of Gramnegative bacilli from positive FAN BacT/ALERT blood culture bottles. Clin. Microbiol. Infect. 16, 137–140. Ramani, R., Chaturvedi, V., 2000. Flow cytometry antifungal susceptibility testing of pathogenic yeasts other than Candida albicans and comparison with the NCCLS broth microdilution test. Antimicrob. Agents Chemother. 44, 2752–2758. Rossney, A.S., Herra, C.M., Brennan, G.I., Morgan, P.M., O'Connell, B., 2008. Evaluation of the Xpert methicillin-resistant Staphylococcus aureus (MRSA) assay using the GeneXpert real-time PCR platform for rapid detection of MRSA from screening specimens. J. Clin. Microbiol. 46, 3285–3290. Schubert, S., Weinert, K., Wagner, C., Gunzl, B., Wieser, A., Maier, T., Kostrzewa, M., 2011. Novel, improved sample preparation for rapid, direct identification from positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. J. Mol. Diagn. 13, 701–706. Shrestha, N.K., Scalera, N.M., Wilson, D.A., Procop, G.W., 2011. Rapid differentiation of methicillin-resistant and methicillin-susceptible Staphylococcus aureus by flow cytometry after brief antibiotic exposure. J. Clin. Microbiol. 49, 2116–2120. Sparbier, K., Schubert, S., Weller, U., Boogen, C., Kostrzewa, M., 2012. Matrix-assisted laser desorption ionization-time of flight mass spectrometry-based functional assay for rapid detection of resistance against beta-lactam antibiotics. J. Clin. Microbiol. 50, 927–937. van der Zwet, W.C., Hessels, J., Canbolat, F., Deckers, M.M., 2010. Evaluation of the Sysmex UF-1000i(R) urine flow cytometer in the diagnostic work-up of suspected urinary tract infection in a Dutch general hospital. Clin. Chem. Lab. Med. 48, 1765–1771.