22: Automated and Manual Systems for Antimicrobial Susceptibility Testing of Bacteria

Automated and Manual Systems for Antimicrobial Susceptibility Testing of Bacteria

Alan T. Evangelista1 and James A. Karlowsky2

1 Drexel University College of Medicine, Philadelphia, PA, USA

2 University of Manitoba College of Medicine, Winnipeg, MB, Canada

22.1 Introduction

A key function of the clinical microbiology laboratory is to perform antimicrobial susceptibility testing (AST) on clinically significant bacterial isolates. Studies have shown that performing an AST method in a timely manner has the potential to improve patient outcomes by the rapid detection of resistance and by the subsequent selection of appropriately targeted antimicrobial agents [3,23,26]. Clinical microbiology laboratories in the United States, Canada, and Europe primarily use commercial broth microdilution testing methods for AST in an automated or semiautomated format [38] for improved test turnaround time. A few of the commercial broth microdilution panels also have the option to be set up and read manually. Since the broth microdilution panels have a fixed configuration of antimicrobial agents and are approved by the US Food and Drug Administration (FDA) for specific microorganism groups, the testing of additional antimicrobial agents or slower growing bacteria is often performed with supplemental agar-based manual AST methods, which include gradient diffusion and disk diffusion. All AST methods provide the qualitative interpretive category results of susceptible, intermediate, and resistant, and the broth microdilution and gradient diffusion methods also provide quantitative minimal inhibitory concentration (MIC) results.

Detailed procedural descriptions of broth microdilution, agar gradient diffusion, and agar disk diffusion methods are not presented in this chapter, and can be found in the Manual of Clinical Microbiology, 9th edn [41,58], in the Clinical and Laboratory Standards Institute (CLSI) documents for antimicrobial susceptibility testing [18–21], and in a review by Jorgensen and Ferraro [38]. In the United States both the CLSI and the FDA set antimicrobial MIC breakpoints for susceptible, intermediate, and resistant interpretive criteria, and in Europe the MIC breakpoints are set by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [25]. The criteria for establishing breakpoints differ slightly among, CLSI, FDA, and EUCAST and there may be minor MIC breakpoint dilution differences and minor inhibition zone diameter differences when comparing the three standards. Both CLSI and EUCAST documents for the interpretive criteria for MICs of relevant antimicrobial agents are reviewed and updated frequently, usually once per year. Commercial AST systems in the United States must use FDA breakpoints. There is a process in place by which the CLSI may periodically submit updated data to the FDA for review with the intention of aligning CLSI and FDA breakpoints for selected antimicrobial–organism combinations. If a regulatory authority such as the FDA changes breakpoints, then a commercial device manufacturer may be required to submit updated data to the FDA for review and approval, which would require 1–3 years for an interpretive breakpoint change to be implemented by a commercial AST system. In the United States either FDA or CLSI susceptibility interpretive breakpoints are acceptable to clinical laboratory accrediting bodies [21].

Commercial systems for AST can be divided into automated, semiautomated, and manual. An automated testing system consists of automated inoculation of MIC panels followed by computer-assisted incubation with reading, interpretation, and reporting functions that do not require manual intervention. A semiautomated system consists of manual or automated inoculation of panels and manual off-line incubation of panels. Each panel is then loaded into an automated reader, and computer-assisted reading and interpretive reporting of MICs is performed. Manual AST panels are inoculated and incubated manually, read visually by laboratory personnel, and the results are either recorded by hand or manually entered into a computer for interpretation and reporting. Each automated, semiautomated, or manual broth microdilution system provides a selection of AST panels with a fixed configuration of antimicrobial agents. Upon request, several companies will prepare custom AST panels for clinical or investigational use.

It is recognized that susceptibility testing methods and panels are subject to periodic modifications and that new systems and panels are introduced into various global markets as an ongoing process. The purpose of this chapter is to review currently used commercial systems, and any omission of a specific method or published evaluation is unintentional. Older published evaluations are reviewed in the first edition of this text.

22.2 Evaluation of commercial AST performance

The evaluation of AST systems is based on guidelines established by the US Food and Drug Administration [71]. Error rates for commercial AST panels are determined by comparing AST results with the reference CLSI broth dilution method using at least 100 unselected clinical isolates of a single genus or species. The detection rate for very major errors (false susceptibility) should be < 1.5% of the test isolates, and major errors (false resistance) should be < 3% of the test isolates. Essential agreement with the reference method (±1 dilution) should be ≥ 90% and categorical agreement with the reference method (same susceptible (S), intermediate (I), resistant ® category result) should be ≥ 90% for each antimicrobial agent and organism combination.

22.3 Automated broth microdilution AST systems

The automated broth microdilution AST systems currently available in the United States include the Vitek 2 (bioMérieux Inc., Durham, NC), the MicroScan WalkAway plus (Beckman Coulter, Inc., Brea, CA), the Phoenix System (BD Diagnostics, Sparks, MD), and the Sensititre ARIS 2X (Thermo Scientific, Lenexa, KS). All four systems provide periodic software and panel upgrades, with most test panels showing good overall essential agreement with the CLSI reference broth dilution method. A listing of the four automated systems and their respective AST panels is presented in Table 22.1.

Table 22.1 Comparison of automated AST systems

Item Vitek 2 (bioMérieux, Inc.) MicroScan WalkAway plus (Beckman Coulter, Inc.) Phoenix (BD Diagnostics) Sensititre ARIS 2X (Thermo Scientific)
Number of wells per panel 64 96 136 96
Number of drugs/GN panel 16–18 on GN panels/cards 21–23 on Neg Combo panels 18–21 on NMIC/ID panels 21–24 on GN MIC panels

19 on GN extension card, XN06 19–24 on Neg/Urine Combo 18–21 on NMIC panels 23 on NF MIC panel

35 for 2 GN cards (GN69+XN06) 22–29 on Neg BP Combo panels
9 on GNUR MIC panel

26 on Neg MIC panel
11 on ESBL plus panel
28 on Emerge NMIC-140 panel 14 on ESBL panel
Number of Gram-negative panels 27 20 20 7
GN AST panel designations GN66-GN93 Neg Combo 31, 32, 44, 50 NMIC/ID121-124, 126-130, 132–134, 139 GN2F–4F, GNX2F

Extension card: XN06 Neg/Urine Combo 33, 35, 45, 46, 51, 60–62 NF

NMIC 121, 123, 124, 126, 129, 133 GNUR2F

Neg BP Combo 30,34, 41, 44, 47 ESB1F

Neg MIC 38; ESBL plus Emerge NMIC-140 panel
ESBL confirmation Yes, on 18 panels Yes, on 15 panels Yes, on 20 panels Yes, on 1 panel, ESB1F
GN urine panels (with nitrofurantoin) Yes, on 24 panels Yes, on 15 panels; Yes, on 20 panels Yes, on 3 panels

9 Neg/Urine Combo panels
GN panels for Pseudomonas spp. and nonfermenters (with pip/tazo, ceftaz or cefep & carbapenem) Yes, on 23 panels Yes, on 19 panels Yes, on 17 panels Yes, on 6 panels
Lower GN CLSI breakpoints:
for cephalosporins and
for carbapenems (CLSI 2010)
Yes, on 27 panels
Yes, on 27 panels
Yes, on 1 panel, ESBL plus
Yes, on 8 panels
Yes, on 2 panels
Yes, on 20 panels
Yes, on 6 panels
Yes, on 6 panels
Number of Gram-positive panels 2 10 11 3
GP panel designations for Staphylococcus spp, Enterococcus spp, Group B Strep GP75 Pos Combo 20, 21, 29, 33, 34
Pos BP Combo 20, 23
Pos MIC 26, 29
PMIC/ID 105–109
PMIC 106–109
S. pneumoniae AST panel GP74 MICroSTREP plus SMIC/ID-101, SMIC-101 STP6F
Data Management Software Observa LabPro Information Manager BDXpert Sensititre Windows (SWIN)
Expert System Advanced Expert System (AES) LabPro AlertEX System Advanced BDXpert SWIN

GN, Gram-negative; GP, Gram-positive; BP, breakpoint; Neg, negative; Pos, positive; pip/tazo, piperacillin/tazobactam; ceftaz, ceftazidime; cefep, cefepime; CLSI, Clinical and Laboratory Standards Institute.

The automated and semiautomated systems discussed in this chapter have the capability of producing standardized or customized patient test reports generated by computer software packages referred to as data management systems. These software systems usually contain an epidemiology component which can archive results, thereby providing specialized reports such as summary reports, infection prevention reports, organism trending reports, and hospital antibiograms, or formatted reports of cumulative susceptibility data. To optimize the availability and transcription accuracy of rapid patient reports, the integrated data from automated and semiautomated systems may be transferred through a computer interface to a laboratory information system (LIS). Specialized reports may then be generated by the LIS in addition to reports generated by the automated system’s software.

An additional level of software enhancement in automated systems is referred to as “expert software,” which examines and validates the antimicrobial susceptibility profile or phenotype of an individual isolate. These expert systems use specific rules or algorithms (preprogrammed or user defined) to flag unlikely resistance patterns and recommended changes. Expert software may also predict cross-resistance to other antimicrobial agents and can facilitate the addition of footnotes or comments to a patient’s report regarding the resistance pattern. An additional advantage of some of the automated systems is a software package that enables interfacing with a pharmacy system so that the microbiology results can be matched with the patient’s record of antimicrobial therapy. Thus, the early detection of in vitro resistance could potentially affect patient outcome if the pharmacy system was alerted to the need to modify therapy. The names of the expert systems available for the automated systems are listed in Table 22.1. A detailed review of evaluations of expert systems for the Vitek 2, MicroScan, and Phoenix systems has been published by Winstanley and Courvalin [76].

The relationship between an automated rapid susceptibility report and the clinical and economic impact on patient care has been the subject of several investigations [3,23,26,39,55,70]. While empiric therapy is commonly initiated in a significant number of hospitalized patients, a physician’s modification to a more appropriate therapy in response to rapid susceptibility reports has been recorded for about 10–20% of cases [23,26]. Modification of therapy may result in a direct cost saving to the hospital [23,26] and lower mortality rates [23]. Thus, automated systems have the potential to offer improved patient care in addition to labor savings, standardization of testing, reproducibility, and data management.

Along with the advantages of automated systems, the purchaser should also be mindful of the limitations of each system. It has been common practice with commercial systems over the past two decades that following the publication and identification of a selective limitation with a pathogen and corresponding antimicrobial agent in an in vitro test panel, the manufacturer often voluntarily removes the panel from the market. An updated panel and software is then often reintroduced after the completion of additional clinical testing and FDA clearance of the new panel. In this chapter, each of the four current automated systems is discussed individually and evaluations (advantages and limitations) are presented as an overview of the current literature.

22.3.1 Vitek 2

The Vitek 2 instrument is a broth microdilution-based AST system (Figure 22.1), which uses 64-well plastic cards containing 17-20 antimicrobial agents (Figure 22.2). Combinations of primary Gram-negative susceptibility cards can be combined with extension susceptibility cards resulting in the testing of 18 or 19 additional antimicrobial agents on the same isolate. The Vitek 2 measures changes in turbidity over time (growth curve), comparing a growth control well with wells containing various drug concentrations. Results are reported in 4–18 h as MIC and S-I-R category, depending on the growth rate and susceptibility parameters of the organism. The Vitek 2 instrument replaced the prior less automated Vitek 1 (Vitek Legacy) instrument over the period 1999–2010. Two Vitek 2 instruments are available with test card (ID and AST) capacities of 60 cards (Vitek 2) and 120 cards (Vitek 2 XL) with an expanded capability to connect two Vitek 2 modules to the same computer. In addition, three Vitek 2 Compact instruments are available with test card capacities of 15, 30, and 60 cards. Table 22.1 lists a variety of susceptibility cards, which are available for Gram-negative and Gram-positive organisms.

Photo of Vitek 2 system.

Figure 22.1 Vitek 2 system.

(Courtesy of bioMérieux.)

Photo of Vitek 2 Gram‐negative ID and AST cards.

Figure 22.2 Vitek 2 Gram-negative ID and AST cards.

(Courtesy of bioMérieux.)

The Vitek 2 system contains computer software to also deduce the susceptibility results for selected Gram-positive bacteria (Staphylococcus spp., Enterococcus spp., Streptococcus agalactiae) for an additional four to ten antimicrobial agents, resulting in the reporting of 23–30 antimicrobial agents per card (Table 22.1). The Vitek 2 system incorporates test setup and sample verification with a Smart Carrier component. All AST inoculum dilutions are performed by the instrument, as well as card sealing and incubator loading functions. Optical reading of cards is performed every 15 min in the Vitek 2, with a multichannel fluorometer and photometer to record fluorescence, turbidity, and colorimetric signals. Susceptibility results are reported in 4–18 h, depending on the organism and susceptibility parameters. The data management system of the Vitek 2 is a PC Windows-based platform termed PC/Observa (Table 22.1). The software is FDA-cleared and is configured by the manufacturer to FDA-defined MIC interpretive breakpoints, as are all automated AST systems. However, the Vitek software also gives the customer the option to configure MIC interpretive breakpoints according to the most recent CLSI AST performance standards [21]. The PC/Observa platform also provides a selection of quality control options, and has multi-instrument access capabilities. The detection of antimicrobial resistance is also facilitated in the Vitek 2 by the Advanced Expert System (AES). This software system validates MIC results by a set of in vitro testing rules based on 20,000 MIC distributions with over 2000 phenotypes, provides result interpretations and corrections, and adds footnotes (CLSI or laboratory defined) for communication to physicians. A biological correction is recommended by the Vitek 2 AES if it detects a single MIC inconsistency with the bacterial identification compared to the internal database. The Vitek 2 system for AST can be connected with the automated microbial identification system, Vitek MS, which uses mass spectrometry technology termed MALDI-ToF (matrix assisted laser desorption ionization time of flight) to provide culture isolate identification results in minutes. The AST and ID systems are connected via the laboratory informatics system, the Vitek Myla.

In an evaluation by Barry et al., MIC values from the Vitek 2 with AES was compared to a reference MIC agar dilution method against clinical isolates tested in five laboratories [4]. The essential agreement (MIC within one dilution of the reference method) for the Vitek 2 was 95.5% for 3074 Gram-negative rods, 96.7% for 1674 staphylococci, and 96.5% for 315 enterococci. In the same study, the Vitek 2 with AES appropriately detected a subset of characterized resistance mechanisms among the Gram-negative rods, staphylococci, and enterococci [4]. Blondel-Hill et al. evaluated the Vitek 2 with AES in comparison to a reference broth microdilution method against 300 Enterobacteriaceae and reported an overall correlation of susceptibility interpretations of 96.2% [8].

Performance characteristics for the Vitek 2 were compared with the Phoenix system (BD Diagnostics) in a study by Eigner et al. with an evaluation of 307 clinical isolates including Enterobacteriaceae, Gram-negative non-fermenters, Staphylococcus spp., and Enterococcus spp. [24]. The overall category agreement for both instruments was 97.0% compared to a reference broth microdilution method, and the minor error, major error (false positive), and very major error (false negative) rates for the Vitek 2 were 2.8, 0.2, and 1.7, respectively. The Vitek 2 with AES was evaluated for its ability to identify β-lactam phenotypes in a test panel of 196 isolates of Enterobacteriaceae and P. aeruginosa, and overall, the AES was able to ascertain the β-lactam phenotype for 93.4% of the isolates tested [62].

The Vitek 2 was evaluated along with five other commercial MIC systems for the accuracy of detecting vancomycin MICs with a range of ≤ 1–8 mg/mL among 129 Staphylococcus aureus isolates from the CDC strain collection [67]. The essential agreement (±1 dilution) for the Vitek 2 GP61 card (currently replaced by the GP75 card, Table 22.1) was 100% and the overall category agreement was 90.7% [67]. Junkins et al. evaluated the implementation of cefoxitin on the Vitek 2 GP66 card to enhance the detection of methicillin-resistant S. aureus (MRSA) in comparison to two reference methods: oxacillin MIC reference broth microdilution and mecA gene detection [42]. For the 448 MRSA and 172 methicillin-susceptible S. aureus tested, the categorical agreement for the Vitek 2 was 99.7%. In a study of susceptibility testing of enterococci, the Vitek 2 was evaluated with 150 clinical isolates of enterococci, which included vanA, vanB, and vanC strains and six species of enterococci [29]. The essential agreement results for ampicillin, vancomycin, teicoplanin, and high-level gentamicin resistance were 93, 95, 97, and 97%, respectively. Kobayashi et al. evaluated the Vitek 2 against 32 strains of vancomycin-resistant enterococci (VRE), which were characterized by PCR as 23 vanA and 9 vanB [45]. In addition to the PCR analysis the Vitek 2 results were compared with two CLSI reference methods, agar dilution and broth microdilution. Among the seven antimicrobial agents tested, the Vitek 2 exhibited one very major error for teicoplanin, no major errors to any of the antimicrobials tested, and seven minor errors for teicoplanin. Overall, 54% of MIC test results were completed by the Vitek 2 within 7 h and all MIC values were determined within 13 h of incubation [45]. The Vitek 2 is capable of performing susceptibility testing of Streptococcus pneumoniae with a configured MIC card (GP74, Table 22.1) containing enriched growth medium. In a report by Jorgensen et al., 53 challenge strains of pneumococci with known resistance mechanisms and 818 clinical isolates were studied with the Vitek 2 compared with CLSI reference broth microdilution method in three separate laboratories [37]. The overall agreement of the Vitek 2 MIC values with the 10 antimicrobial agents studied was 96.3% with a mean time of results generated of 8.1 h.

22.3.2 MicroScan WalkAwayplus System

The MicroScan WalkAway plus System (Figure 22.3) automated AST system is a broth microdilution method utilizing a standard 96-microwell panel (Figure 22.4). The WalkAway plus System is available in both 40- and 96-panel capacity models and was designed to replace the WalkAway-40 and the WalkAway 96 SI models. The instrument is capable of reading either conventional or rapid panels. The microwells contain serial dilutions of dehydrated antimicrobial agents. Three types of panel configurations for Gram-negative and three for Gram-positive bacteria are available: (i) Combo panels containing both antimicrobial dilutions and identification (ID) substrates; (ii) Breakpoint Combo panels containing antimicrobials in a breakpoint dilutions and ID substrates; and (iii) MIC panels containing only antimicrobial dilutions. Breakpoint panels contain only two or three concentrations of each antimicrobial agent, and the resulting MIC corresponds to the interpretive category S, I, or R. In contrast, standard MIC panels contain a wide range of doubling dilution antimicrobial concentrations for the determination of the MIC. The MicroScan panel types are listed in Table 22.1. The panels are manually inoculated and rehydrated by using the RENOK inoculator and then placed into the incubator-reader component of the WalkAway. Results are obtained after 15 to 18 h by turbidimetric readings of overnight conventional panels and after 4.5–18 h readings of rapid panels. Combination Gram-negative ID and AST panels called MicroScan Synergies plus Negative and rapid/S plus Negative panels were introduced in 2005, voluntarily recalled in 2013, and no longer available in the United States. The Synergies plus Negative 96-well panels were configured with 36 wells containing fluorogenic substrates for rapid organism identification (2.5 h) and 60 wells with antimicrobials in Mueller–Hinton broth for the turbidimetric determination of MICs utilizing a proprietary technique to optimize growth and allow readings from 4.5 to 18 h when needed. Since the rapid panels were no longer available at the time of writing this chapter, only the conventional panels are listed in Table 22.1. The WalkAway plus System includes the LabPro Information Manager system software for ID and AST data management. The optional LabPro Alert System software incorporates the detection of unusual resistance results and incorporates institution-specific infection prevention guidance into footnotes or comments for physician review. An expert software package was added in version 3 of the LabPro AlertEX System [76].

Photo of MicroScan WalkAway plus system.

Figure 22.3 MicroScan WalkAway plus system.

(© 2015 Beckman Coulter, Inc., all rights reserved, used with permission.)

Photo of MicroScan Combo ID and AST panels.

Figure 22.4 MicroScan Combo ID and AST panels.

(© 2015 Beckman Coulter, Inc., all rights reserved, used with permission.)

An evaluation of the MicroScan WalkAway system was conducted by Rittenhouse et al., with results from a conventional MicroScan Gram-negative MIC panel compared to reference broth microdilution using 500 Gram-negative clinical isolates tested against nine antimicrobial agents [60]. Very major errors (false susceptibility) were detected at a rate of 3.0% (15 of 500), and no major errors (false resistance) were detected. Jorgensen et al. evaluated the MicroScan ESBL plus panel against 100 genetically characterized extended-spectrum β-lactamase (ESBL)-producing isolates consisting of 64 Enterobacteriaceae that produced CTX-M, 17 that produced SHV or TEM ESBL, and 19 that produced CTX-M and SHV ESBLs [40]. The MicroScan ESBL plus panel detected 98% of the isolates. A study by Bulik et al. compared MicroScan panels and reference broth microdilution in an evaluation of meropenem MICs from carbapenemase-producing Klebsiella pneumoniae with KPC genotypes [14]. A total of 46 K. pneumoniae isolates were tested, which were all modified Hodge test positive and blaKPC positive. The MicroScan panels demonstrated a 95.6% essential agreement with the reference method, with 2.2% minor errors and no major or very major errors [14].

MicroScan Pos MIC conventional (overnight) panels were evaluated by Tenover et al. for the ability to detect linezolid susceptibility against a challenge panel of 50 enterococci and 50 staphylococci [69]. The challenge panel included 17 enterococci and 15 staphylococci that were nonsusceptible to linezolid. The MicroScan Pos MIC panels were read using the WalkAway with LabPro software and the category agreement with the CLSI broth microdilution reference method was 96.0%. In a study by Swenson et al., 129 S. aureus isolates were evaluated for the ability of the MicroScan Pos MIC 26 panel to detect vancomycin susceptible (n = 84) and vancomycin intermediate (n = 45) strains from the CDC collection [67]. The MicroScan Pos MIC 26 panel demonstrated an overall category agreement of 92.2% identifying all 45 of the VISA strains and categorizing 10 of the 84 vancomycin-susceptible strains as VISA. MicroScan has a specific panel for the susceptibility testing of Streptococcus pneumoniae called the MICroSTREP plus panel (Table 22.1). In a report by Mittman et al., 311 clinical isolates of S. pneumoniae were tested using the MICroSTREP plus panel compared to a reference broth microdilution method [52]. The overall categorical agreement of the MicroScan MIC values with the 13 antimicrobial agents studied was 99.5% and the categorical agreement for penicillin was 98.7%.

22.3.3 Phoenix System

The BD Phoenix Automated Microbiology System utilizes combination panels for bacterial ID and AST. The combination MIC/ID panels contain a total of 136 wells, of which 51 are utilized for ID and up to 85 are utilized for AST (Table 22.1). A bacterial inoculum for ID and a second inoculum for AST are poured into receptacles of the respective ID and AST sections of the single combination panel. The panels are tilted and each dehydrated well is then self-inoculated. Susceptibility results are determined in the Phoenix system by utilizing a resazurin-based redox dye as well as kinetic measurements of turbidity to detect bacterial growth in the presence of an antimicrobial agent. Up to 100 panels can be placed into the Phoenix system for ID, AST, or combination of ID and AST. The panels are automatically incubated for up to 18 h, and the results are read and reported. The addition of a new Gram-negative susceptibility panel in 2014 called the Emerge NMIC-140 (Table 22.1) was designed for MIC susceptibility testing on the Phoenix system to be used in combination with the BD Bruker MALDI-ToF Biotyper system for the rapid identification of culture isolates using mass spectrometry. The two systems are connected through the BD EpiCenter software system for data management. The Phoenix system is shown in Figure 22.5, and the Phoenix Combo ID/AST panels are show in Figure 22.6.

Photo of Phoenix system.

Figure 22.5 Phoenix system.

(Courtesy and © 2015 Becton, Dickinson and Company.)

Photo of Phoenix ID/AST panels.

Figure 22.6 Phoenix ID/AST panels.

(Courtesy and © Becton, Dickinson and Company.)

An evaluation of the Phoenix system was performed by Eigner et al. with an evaluation of 307 clinical isolates including 141 Enterobacteriaceae, 22 Gram-negative non-fermenters, 93 Staphylococcus spp., and 51 Enterococcus spp. [24]. The category agreement for the Phoenix system compared to a reference MIC broth microdilution method was 96.5% for Enterobacteriaceae, 84.8% for Gram-negative non-fermenters, 98.9% for Staphylococcus spp., and 98.0% for Enterococcus spp. The overall category agreement was 97.0% with 3.0% minor errors, 0.3% major errors (false positives), and 0.6% very major errors (false negatives). The evaluation was performed using NMIC/ID 14 panels for Gram-negative rods and PMIC/ID 13 panels for Gram-positive cocci, which have since been replaced and updated on the BD Phoenix system (Table 22.1).

Junkins et al. evaluated the implementation of cefoxitin on the Phoenix PMIC/ID-102 panel to determine if the detection of methicillin-resistant S. aureus (MRSA) was enhanced in comparison to two reference methods: oxacillin MIC reference broth microdilution and mecA gene detection [42]. For the 448 MRSA and 172 methicillin-susceptible S. aureus tested, the categorical agreement for the Phoenix was 99.8%. The Phoenix PMIC/ID-100 panel was evaluated by Tenover et al. for the ability to detect linezolid susceptibility against a challenge panel of 50 enterococci and 50 staphylococci [69]. The challenge panel included 17 enterococci and 15 staphylococci that were nonsusceptible to linezolid. The overall categorical agreement for the Phoenix panel compared to the CLSI broth microdilution reference method was 89.6% and the essential agreement (+/– one MIC doubling dilution) was 95.8%. Buchan et al. evaluated the ability of the Phoenix system to detect inducible resistance to clindamycin among 524 clinical isolates of Staphylococcus spp. [11]. Compared to a double-disk diffusion reference method to detect inducible clindamycin resistance, the Phoenix showed a sensitivity of 100% and specificity of 99.6%.

Richter et al. conducted a multicenter evaluation of the Phoenix system for susceptibility testing of 13 agents against 2,013 streptococcal isolates, including 938 S. pneumoniae, 396 group B streptococci, 369 viridans group streptococci, 290 β-hemolytic Streptococcus groups A, C, and G, and 20 other streptococci [59]. In comparison to a broth microdilution reference method, the categorical agreement of the Phoenix panels ranged from 92 to 100% with one exception for viridians streptococci and penicillin, which was 87%. Mittman et al. evaluated 311 clinical isolates of S. pneumoniae using the Phoenix SMIC/ID-100 panel compared to a reference broth microdilution method [52]. The overall categorical agreement of the Phoenix panel with the 13 antimicrobial agents studied was 99.3% and the categorical agreement for penicillin was 95.5%.

22.3.4 Sensititre ARIS 2X

Sensititre ARIS (Automated Reading and Incubation System) 2X is an automated, bench-top incubation and reading instrument that accommodates up to 64 Sensititre 96-microwell panels for broth microdilution antimicrobial susceptibility testing and/or organism identification. Sensititre antimicrobial susceptibility testing panels for diagnostic use contain serial dilutions of dehydrated antimicrobial agents and are intended to provide a MIC (μg/mL) and MIC interpretation (susceptible, intermediate, resistant). Panel configurations are available for Gram-positive and Gram-negative bacteria and are listed in Table 22.1; some antimicrobial agents are only tested at MIC breakpoint concentrations. In addition to the commercial panels listed in Table 22.1, Thermo Scientific (previously distributed by Trek Diagnostics Systems) can manufacture custom MIC panels (dehydrated or frozen) for investigators performing surveillance studies and evaluations of new antimicrobials. Sensititre panel inoculation may be automated, using the Sensititre AIM (Automated Inoculation Delivery System), or performed manually using a Sensititre Electronic Pipette. The inoculated panels are then loaded manually into the Sensititre ARIS 2X. Antimicrobial susceptibility testing panels for diagnostic use employ a proprietary substrate (non-fluorescent) in the media used to suspend the bacterial isolate and perform panel inoculation. Hydrolysis of the substrate by bacterial enzymes releases fluorescence which is used to monitor growth in each well and determine MIC endpoints; a computer algorithm converts the fluorescent signal to a MIC. The Sensititre ARIS 2X uses an internal bar code scanner to identify each panel type, then it assigns an appropriate incubation time, and when this assigned time has elapsed (16 to 24 h), the system transports the panel to the OptiRead Automated Fluorometric Plate Reading System within the ARIS 2X with no manual intervention. The Sensititre Windows (SWIN) software system offers automated expert analysis, semi-automated analysis, or manual options on a single software platform. The current Sensititre ARIS 2X instrument was released in 2004 and is shown in Figure 22.7 and the Sensititre panels are shown in Figure 22.8.

Two photos of Thermo Scientific™ Sensititre™ ARIS™ 2X ID/AST System.

Figure 22.7

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Dec 10, 2017 | Posted by in MICROBIOLOGY | Comments Off on 22: Automated and Manual Systems for Antimicrobial Susceptibility Testing of Bacteria

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