First Comprehensive Evaluation of the M I C Evaluator Device Compared to Etest and CLSI Broth Microdilution for MIC Testing of Aerobic Gram Positive and Gram Negative Bacterial Species

(1)

Compared to Etest and CLSI Broth Microdilution for MIC Testing of

Aerobic Gram-Positive and Gram-Negative Bacterial Species

R. P. Rennie,a_{L. Turnbull,}a_{C. Brosnikoff,}a_{and J. Cloke}b

Medical Microbiology Research Laboratory, Department of Laboratory Medicine and Pathology, University of Alberta Hospital, Edmonton, Alberta, Canada,a

and Thermo Fisher Scientific, Basingstoke, United Kingdomb

The M.I.C. Evaluator strip (Thermo Fisher Scientific, Basingstoke, United Kingdom) uses a methodology similar to that of Etest.

In this first assessment of the M.I.C. Evaluator device, 409 strains of aerobic Gram-positive bacteria (staphylococci, streptococci,

and enterococci) and 325 strains of

Enterobacteriaceae

,

Pseudomonas

species, and

Acinetobacter

species were tested by M.I.C.

Evaluator strip, Etest, and broth microdilution as a reference standard. The Gram-positive bacteria included staphylococci

(me-thicillin-resistant

Staphylococcus aureus

, methicillin-susceptible

S. aureus

, and coagulase-negative staphylococci),

Streptococcus

pneumoniae

, beta-hemolytic streptococci and viridians group strains, vancomycin-resistant enterococci, and other enterococci.

The Gram-negative bacteria included 250 strains of 60

Enterobacteriaceae

species plus 50

Pseudomonas

and 25

Acinetobacter

species. A total of 14 antimicrobial agents (depending on the species) were included. The same methodology and reading format

were used for M.I.C. Evaluator strips and Etest. Broth microdilution methodology was performed according to CLSI document

M07-A8. For the clinical strains,

>

95% of results were plus or minus one doubling dilution for all species. There were fewer than

5% minor errors, fewer than 3% major errors, and fewer than 1% very major errors. M.I.C. Evaluator strips and Etest often

re-ported higher MICs than the reference broth microdilution method. The M.I.C. Evaluator strips provided results comparable to

those of the predicate Etest device and are of value for the accurate testing of MICs for these important pathogens.

T

here are three methods that permit accurate MIC testing.

These are broth and agar dilution tests and a gradient endpoint

method that incorporates the antimicrobial agent as a defined

gradient onto the back of a solid plastic strip. MICs are read where

the zone of inhibition intersects with the drug concentration on

the strip. Until now, the only gradient endpoint device available

was Etest, developed by AB Biodisk, Solna, Sweden, and currently

marketed by bioMérieux. This method has previously been shown

to be an accurate and simple alternative to reference broth or disk

methods (9, 12, 15, 18).

The M.I.C. Evaluator strip (M.I.C.E) is a new gradient

end-point susceptibility test device. Both the M.I.C. Evaluator strip

and Etest are performed similarly and have excellent utility for

directed MIC testing against specific agents and microorganisms

isolated from clinical samples. The advantage of these devices is

the ease of use and accurate MIC results for patient management.

MIC data also are valuable for clinical pharmacists to determine

optimal pharmacodynamic dosing. In these situations, it is

impor-tant to be able to detect resistance early in the course of treatment

(5, 7, 10). To ensure that any new device is effective in the clinical

laboratory, appropriate validation studies, including both control

strains and an extensive collection of clinical isolates, is essential to

ensure evaluable results for patient care.

Gram-positive and Gram-negative aerobic and facultative

an-aerobic bacteria occupy the largest group of microorganisms

as-sociated with infections in patients in critical care settings. The

consequences of inappropriate therapy or of inadequate dosing in

these patients may have important clinical consequences. It is,

however, important that the

in vitro

MIC device provides an

ac-curate estimation of the efficacy of the antimicrobial agent against

the infecting organism. We compared the new M.I.C. Evaluator

strip to Etest to investigate any discordance between the two

de-vices, as well as to the standard CLSI reference broth

microdilu-tion method (2), on a collecmicrodilu-tion of staphylococci, streptococci,

and enterococci. We also evaluated the device on a large collection

of enterobacteriaceae, as well as

Pseudomonas

species and

Acineto-bacter

species. This is the first comprehensive evaluation of this

new gradient endpoint device that compares it to the CLSI

refer-ence broth dilution method. The M.I.C. Evaluator device has been

compared to the British Society for Antimicrobial Chemotherapy

(BSAC) method, with observations similar to those in the current

study (1, 8). Other collections of bacterial species, including

an-aerobes and fastidious bacteria, are reported in a companion

pub-lication (11).

MATERIALS AND METHODS

Bacterial species. (i) Gram-positive bacteria.A total of 409 fresh or re-cently frozen clinical strains of staphylococci, streptococci, and entero-cocci were tested. All isolates were from individual patients at the Univer-sity of Alberta Hospital, Edmonton, Canada. They were identified in our laboratory using primary tests, standard automated methods, or molecu-lar tests as required. Strains were stored at⫺70°C in litmus milk prior to testing. Strains were also characterized for resistance determinants where appropriate. Methicillin resistance in Staphylococcus aureus was con-firmed by cefoxitin disc testing, PBP 2=testing, andmecAgene testing. There were 157 strains ofS. aureus, including methicillin-susceptible

Received16 September 2011Returned for modification3 October 2011 Accepted3 January 2012

Published ahead of print11 January 2012

Address correspondence to R. P. Rennie, [email protected].

doi:10.1128/JCM.05395-11

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strains (MSSA) (84), methicillin-resistantS. aureus(MRSA) (20), and coagulase-negative staphylococci (CNS) (53); 151 strains of streptococci, including Streptococcus pneumoniae(60), beta-hemolytic streptococci (40), and viridans group streptococci (51); and 100 strains of enterococci (52 vancomycin-resistant enterococci [VRE] and 48Enterococcus spe-cies). The species of strains of coagulase-negative staphylococci and van-comycin-susceptible enterococci were not fully determined, since this did not bear on the comparison of antimicrobial test results. Among the 51 strains included as viridans group streptococci, there were 13S. mitis, 4S. salivarius, 3S. sanguis, 2S. dysgalactiae, 1 each ofS. mutansandS. equi

subsp.zooepidemicus, and 16S. anginosusstrains; the species of 11 strains were not determined. Within the beta-hemolytic streptococci, there were 25 strains ofS. pyogenes(group A), 11 strains ofS. agalactiae(group B), and 4 strains of other species (group C). Among the vancomycin-resistant enterococci, 29 strains carried VanA and 14 strains carried VanB. There were 8 strains classified as VanC (Enterococcus casseliflavusorEnterococcus gallinarum), and there was 1 strain ofE. gallinarumwith both VanA and VanC genes. The quality control strains for Gram-positive tests wereS. aureusATCC 29213,S. pneumoniaeATCC 49619, andEnterococcus faeca-lisATCC 29212.

(ii) Gram-negative bacteria.For the enterobacteriaceae, a total of 250 strains, each from individual patients, were tested. There were 16 com-mon genera and 60 species in this collection of strains (Table 1). For

Pseudomonasand Acinetobacterspecies, the following organisms were tested: 50Pseudomonasspecies (40P. aeruginosa, 5P. stutzeri, 3P. fluore-scens, and 1 each ofP. pseudoalcaligenesandP. mendocina) and 25 strains of theAcinetobacter baumanniicomplex. The same methodologies as those for the Gram-positive species were used for the identification and for the storage of strains prior to testing.

Four standard quality control strains were tested against the Gram-negative isolates as appropriate. These wereEscherichia coliATCC 25922,

P. aeruginosaATCC 27853,S. aureus ATCC 29213, and Enterococcus faecalisATCC 29212. CLSI quality control MIC ranges were available for all antimicrobial agents tested in this study.

Antimicrobial agents. (i) Gram-positive bacteria.A total of 13 anti-microbial agents were tested against the Gram-positive bacterial strains. The agents tested were ampicillin, amoxicillin, amoxicillin-clavulanate, cefotaxime, ciprofloxacin, erythromycin, gentamicin, levofloxacin, lin-ezolid, oxacillin, penicillin, tetracycline, and vancomycin. Agents were tested as appropriate for the species.

(ii) Gram-negative bacteria.In this study, nine agents produced for the M.I.C. Evaluator strips were tested: ampicillin, amoxicillin, amoxicil-lin-clavulanate, cefotaxime, tetracycline, gentamicin, ciprofloxacin, imi-penem, and levofloxacin. The concentration ranges were similar for Etest and the M.I.C. Evaluator device. Etest strips were purchased from the manufacturer (bioMérieux). M.I.C. Evaluator strips were supplied by Thermo Fisher Scientific, Basingstoke, United Kingdom.

CLSI broth microdilution tests.Standard CLSI broth microdilution methodology (2) was used to test for susceptibility, with media of cation-supplemented Mueller-Hinton broth (CSMHB) for staphylo-cocci, enterostaphylo-cocci, and for the Gram-negative species and CSMHB with 5% horse blood for streptococci. Strains were incubated at 35°C for 20 to 24 h, and susceptibility was determined according to standard CLSI breakpoints (3).

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Gradient endpoint device testing.The same strains were tested using the same inocula as those used for the broth microdilution tests. The bacterial strains were inoculated onto appropriate Mueller-Hinton agar (Thermo Fisher, Basingstoke, United Kingdom) according to CLSI M07-A8 (2). M.I.C.E and Etest strips were added to the plates according to the manufacturers’ instructions and were incubated for 20 to 24 h at 35°C. Complete inhibition was read for each manufacturers’ strip at the point where the elliptical zone intersected with the strip. For trimethoprim-sulfamethoxazole, erythromycin, tetracycline, and linezolid, where 80% inhibition was taken as the endpoint, the manufacturer’s diagrams and photographs were used for the analysis of the test results.

TABLE 1Enterobacterial species testeda

Species No. of strains

Escherichia coli 44

Escherichia coli0157:H7 3

Escherichia fergusonii 1

Escherichia hermanii 1

Escherichia vulneris 1

Klebsiella pneumoniae 29

Klebsiella oxytoca 14

Klebsiella ozaenae 4

Klebsiella rhinoscleromatis 2

Enterobacter cloacae 24

Enterobacter aerogenes 15

Enterobacter sakazakii 3

Enterobacter amnigenus 4

Enterobacter agglomerans 1

Enterobacter gergoviae 2

Enterobacter asburiae 2

Enterobacter cancerogenus 1

Salmonella enterica

Serovar Typhi 2

Serovar Enteritidis 2

Serovar Typhimurium 2

Serovar Paratyphi 4

Serovar Heidelberg 1

Serovar Kentucky 1

Serovar Hadar 1

Serovar Newport 1

Serovar Cholerasuis 1

Serovar Arizonae 1

Serovar Muenchen 1

Serovar Derby 1

Serovar Infantis 1

Serovar Lomalinda 1

Citrobacter freundii 11

Citrobacter koseri 3

Citrobacter amalonaticus 1

Citrobacter braakii 1

Citrobacter farmeri 1

Citrobacter sedlackii 1

Citrobacter werkmanii 1

Citrobacter youngae 1

Shigella sonnei 5

Shigella dysenteriae 4

Shigella boydii 5

Shigella flexneri 5

Proteus mirabilis 10

Proteus vulgaris 7

Proteus penneri 3

Yersinia frederiksenii 1

Yersinia intermedia 1

Yersinia enterocolitica 2

Ewingella americana 1

Serratia marcescens 3

Serratia odorifera 1

Serratia liquefaciens 1

Morganella morganii 3

Providencia stuartii 2

Providencia rettgeri 1

Hafnia alvei 1

Ochrobactrum anthropi 1

Pantoea agglomerans 1

Edwardsiella tarda 1

a_{Sixteen genera and 250 strains were tested.}

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Performance criteria.Standard performance criteria were included in this method for comparisons of the two gradient endpoint devices against the reference broth microdilution method, includingⱖ95% quality con-trol strains within acceptable ranges, ⱖ90% essential and categorical agreement,ⱕ10% minor errors,ⱕ3% major errors, andⱕ1.5% very major errors. Essential agreement was defined for this study as a result being plus or minus one doubling dilution of that from broth microdilu-tion. These criteria are identified in FDA guidance documents (16) and are universally used.

RESULTS

The quality control organisms for both positive and

Gram-negative isolates were tested a total of seven times during this

evaluation. Quality control strains were included with each test

run. Results for all of the strains tested showed that the MICs were

within published CLSI ranges for each of the antimicrobial agents

tested. Each strain tested in the study was isolated from a different

patient, although as would be expected in most current hospital

settings, it is possible that some of the resistant strains were in fact

clonal and associated with ongoing outbreaks.

For the Gram-negative enterobacterial isolates, a total of 250

strains of 16 genera and 60 species were tested by all three methods

(Table 1). For isolates of

Pseudomonas

and

Acinetobacter

species, a

total of 75 strains were tested.

The number of susceptible (S), intermediate (I), and resistant

(R) strains and their essential agreements in comparisons of

M.I.C. Evaluator strips and Etest to the standard reference method

for each of the Gram-positive genera are summarized in Tables 2

(staphylococci), 3 (streptococci), and 4 (enterococci). There were

very few resistant streptococci, as would be expected, and S, I, and

R results for those species are not included. The combined error

rates for all Gram-positive bacteria are shown in Table 5.

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For the staphylococci, essential agreement was

⬍

90% for all

agents except erythromycin and gentamicin. Categorical

agree-ment, however, was

⬎

95% for all agents except cefotaxime and

TABLE 2Essential agreement for M.I.C.E and Etest compared to broth microdilution for 157 strains of staphylococci

Drug

No. of strains in each resistance category

No. of strains according to no. of doubling dilutions away from reference

Essential agreement (%) (⫾1 dilution)

⬎⫺2 ⫺2 2 ⬎2

S I R M.I.C.E Etest M.I.C.E Etest M.I.C.E. Etest M.I.C.E Etest M.I.C.E Etest

Cefotaxime 132 9 16 0 0 0 0 105 (67) 128 (82) 31 16 21 13

Ciprofloxacin 104 3 50 0 0 1 1 67 (43) 70 (45 62 55 27 31

Erythromycin 81 0 76 7 6 3 0 147 (94) 150 (96) 0 0 0 1

Gentamicin 133 7 17 3 3 7 8 144 (92) 145 (92) 2 1 1 0

Levofloxacin 102 4 51 0 0 2 2 131 (83) 129 (82) 21 22 3 4

Linezolid 155 1 1 0 23 39 53 116 (74) 81 (52) 2 0 0 0

Oxacillin 111 0 46 0 0 1 1 103 (66) 95 (61) 20 29 34 33

Penicillin 26 0 131 12 17 9 9 110 (70) 110 (70) 20 13 6 8

Tetracycline 142 3 12 1 1 0 0 115 (73) 70 (45) 32 68 9 18

Vancomycin 154 2 1 0 0 0 0 95 (61) 132 (84) 59 9 3 1

Total 1,140 29 401 23 50 62 74 1,133 (72) 1,110 (71) 218 197 83 96

TABLE 3Essential agreement for M.I.C. Evaluator strips (M.I.C.E) and Etest compared to broth microdilution for 151 strains of streptococci

Agent

⬎⫺2 ⫺2 2 ⬎2

M.I.C.E Etest M.I.C.E Etest M.I.C.E. Etest M.I.C.E Etest M.I.C.E Etest

Amoxicillin 1 1 1 3 144 (95) 144 (95) 4 2 1 1

Ampicillin 1 1 6 3 141 (93) 144 (95) 1 1 2 2

Cefotaxime 0 0 1 1 120 (79) 125 (83) 23 20 7 5

Ciprofloxacin 0 0 0 0 136 (90) 139 (91) 13 11 2 1

Erythromycin 1 0 2 1 82 (54) 66 (44) 47 33 19 51

Gentamicin 6 9 4 6 77 (51) 105 (70) 49 24 15 9

Imipenem 2 0 7 2 137 (90) 81 (54) 3 47 2 21

Levofloxacin 0 0 0 0 130 (86) 132 (87) 19 17 2 2

Linezolid 1 1 0 6 139 (92) 142 (94) 11 2 0 0

Oxacillin 1 1 7 2 137 (91) 141 (94) 4 3 2 2

Penicillin 0 0 3 2 133 (88) 137 (90) 13 11 2 1

Tetracycline 1 1 1 1 122 (81) 111 (73) 16 27 11 11

Vancomycin 1 0 1 0 145 (96) 148 (98) 3 2 1 1

Total 15 14 33 27 1,643 (84) 1,615 (82) 206 200 66 105

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oxacillin. M.I.C. Evaluator strips and Etest performed in an

equiv-alent manner. As shown in Table 2, both gradient endpoint

de-vices gave larger numbers of results at two or more dilutions above

the reference method MIC for most of the agents tested with

staphylococci. The major issue that was discovered was with

oxa-cillin and penioxa-cillin. Higher MICs for the two gradient devices

resulted in some major errors for oxacillin, and some lower

gra-dient penicillin MICs resulted in a number of very major errors.

Therefore, the gradient endpoint devices would report oxacillin as

resistant, whereas the reference method called these strains

susceptible. The opposite occurred for penicillin (Table 5). On

closer examination, all of the oxacillin discrepancies were among

methicillin-resistant

S. aureus

isolates that were captured by

M.I.C.E strips and Etest but not by broth microdilution. For

pen-icillin, discordant results all occurred among oxacillin-susceptible

S. aureus

strains. We did not observe any small colonies (so-called

fuzzy zones) surrounding the gradient endpoint strips in these

isolates. There were two very major errors with gentamicin, one

for an

S. pyogenes

isolate and one for an

S. anginosus

isolate.

For the streptococci (Table 3), essential agreement was low for

erythromycin, gentamicin, tetracycline, and cefotaxime, but the

categorical agreement was

⬎

95% for all agents that have CLSI

breakpoints except for erythromycin (90 and 87% for M.I.C.

Eval-uator and Etest, respectively). The essential agreement of Etest

(54%) for imipenem was much lower than that of M.I.C.E (90%).

As in the case of staphylococci, streptococcal MICs with the

gra-dient endpoint devices tended to be higher than the reference

MICs (Table 3). Most of the streptococci have very low MICs for

all agents tested, such that a 2-fold difference in MIC between the

gradient endpoint and reference broth method might only reflect

a change from a MIC of 0.004 to 0.016, for example. This would

not affect categorical agreement or reporting on the likely efficacy

of that agent.

For the 100 strains of enterococci tested, ampicillin had a

high essential agreement, but for linezolid and vancomycin the

agreements were variable and lower between the two devices

compared to reference broth microdilution. For linezolid, all

results were clustered within a narrow range of MICs (0.5 to 4

mg/liter), so that while the essential agreements were good, the

categorical agreements were lower (74 and 70% for M.I.C.

Evaluator and Etest, respectively) because the result for some

strains was 4 mg/liter, whereas the MIC for the gradient

end-points devices was 2 mg/liter. For vancomycin, as was the case

of other Gram-positive species, the gradient endpoint devices

had slightly higher MICs than the reference method. Thus,

almost all of the categorical results had minor errors for both

linezolid and vancomycin. There was one major error for

M.I.C. Evaluator strips and Etest with linezolid and one major

error for M.I.C.E with vancomycin (Table 5).

For the Gram-negative species studied (Table 6), essential

agreement also was high, except for the two fluoroquinolones

cip-rofloxacin and levofloxacin, but these results did not affect

cate-gorical agreements. Catecate-gorical agreement was

ⱖ

95% for all

strains tested except for ampicillin and amoxicillin-clavulanic acid

(both were 90 to 91%). Very major errors were observed only with

ampicillin and amoxicillin-clavulanate (Table 7). All of these

oc-curred with

P. vulgaris

and one

C. freundii

isolate. All other

mi-croorganism-antimicrobial agent combinations were within

ac-ceptable rates of error.

DISCUSSION

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This study was performed with the first available antimicrobial

agents produced for the M.I.C. Evaluator strips. For those agents,

the comparison was made first against Etest as the predicate device

to identify any major method discrepancies and then against the

CLSI broth microdilution method as the reference method.

TABLE 4Essential agreement for M.I.C.E and Etest compared to broth microdilution for 100 enterococcal strains

Agent

No. of strains in each resistance categorya

⬎⫺2 ⫺2 2 ⬎2

S I R M.I.C.E Etest M.I.C.E Etest M.I.C.E Etest M.I.C.E Etest M.I.C.E Etest

Ampicillin 63 0 38 0 0 1 1 92 (91) 95 (94) 8 5 0 0

Gentamicin NA 0 0 0 0 72 (71) 86 (85) 21 14 8 1

Imipenem NA 0 0 2 1 97 (96) 96 (95) 2 3 0 1

Linezolid 70 31 0 1 0 0 10 100 (99) 91 (90) 0 0 0 0

Vancomycin 61 9 31 0 0 1 1 78 (77) 92 (91) 20 6 2 2

Total 194 40 69 1 0 4 13 439 (88) 460 (92) 51 28 10 4

a

NA, no CLSI breakpoints available.

TABLE 5Error rates for 409 strains of staphylococcal, streptococcal, and enterococcal species

Agent

No. (%) of errors

Minor Major Very major

M.I.C.E Etest M.I.C.E Etest M.I.C.E Etest

Amoxicillin 0 0 0 0 0 0

Ampicillin 4 (2) 5 (3) 0 0 0 0

Cefotaxime 20 (6) 15 (5) 12 (4) 9 (3) 0 0 Ciprofloxacin 6 (4) 7 (4) 2 (1) 1 (1) 0 0 Erythromycin 16 (5) 16 (5) 7 (2) 8 (3) 0 0

Gentamicin 9 (6) 5 (3) 0 0 2 (1) 0

Imipenem 2 (1) 1 (1) 0 0 0 0

Levofloxacin 2 (1) 2 (1) 0 0 0 0

Linezolid 27 (7) 31 (8) 1 (1) 1 (1) 0 0 Oxacillin 1 (1) 1 (1) 16 (10) 16 (10) 0 0 Penicillin 6 (2) 4 (1) 1 (1) 0 7 (2) 6 (2) Tetracycline 1 (1) 1 (1) 2 (1) 2 (1) 0 0

Vancomycin 15 (4) 10 (2) 1 (1) 0 0 0

Total 109 (3.3) 98 (2.5) 42 (1.5) 37 (1.4) 9 (0.2) 6 (0.2)

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The data showed that the M.I.C. Evaluator strips are

compara-ble to Etest and to the broth dilution reference method for these

agents against a comprehensive range of aerobic Gram-positive

and Gram-negative bacterial species. For most antimicrobial

agents with most bacterial species, it was remarkable how similarly

the two gradient endpoint methods performed for a large number

of strains of these varied species. Additional agents are now being

produced and will be tested in the same manner to verify the

capabilities of the M.I.C. Evaluator strips.

The categorical agreements for both M.I.C. Evaluator strips

and for Etest were comparable. The most notable issues occurred

with beta-lactam antimicrobials, erythromycin, and with the

fluo-roquinolones. Since all of these agents were not tested against

every species, it is difficult to know if this is a genus phenomenon

or is broadly observed across species. For erythromycin, it is well

known that there is less agreement between broth and agar

meth-ods, since testing is affected by pH changes from the CO

2

required

for incubation in agar. We have also discovered with the

entero-bacteriaceae, for example, that there are more minor errors with

beta-lactam agents. In most cases the gradient endpoint methods

tend to give higher MICs than the reference broth dilution

method, and this was consistent for the Gram-positive species. For

a number of antimicrobial-organism combinations, particularly

carbapenems, the Etest has been reported to yield higher MICs

than broth microdilution (4, 6, 13, 17). It is possible that the small

volume used in the broth method reduces the likelihood of finding

slower-growing resistant subpopulations, but this is not well

un-derstood.

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Unlike our findings with the enterobacteriaceae, categorical

agreements with ampicillin for streptococci and enterococci were

very high. This is due to very low MICs for these species compared

to those for enterobacteriaceae, so the categorical agreements

would be expected to be much better. We have noted that with

some microorganism-antimicrobial agent combinations, both

M.I.C. Evaluator strips and Etest tend to report slightly higher

MICs than broth microdilution and may well identify resistance

determinants more readily (data not shown). This phenomenon

has been observed in comparisons of the Etest to commercial

broth dilution systems when testing for resistance markers such as

vancomycin in staphylococci (14). In this study, we observed

higher oxacillin MICs among the staphylococci, resulting in 10%

major errors for both M.I.C. Evaluator strips and Etest compared

to the reference broth method. We determined that these errors

were for methicillin-resistant staphylococci. This is significant,

since the reference broth dilution method would undercall

oxa-cillin resistance. Most laboratories use more than one test to detect

TABLE 6Combined essential agreement for 325 strains of enterobacterial,Pseudomonas, andAcinetobacterspecies for M.I.C.E and Etest compared to broth microdilution

Agent

No. of strains in each resistance category

Essential agreement (%) (_⫾1 dilution)

⬎⫺2 _⫺2 _⫹2 _⬎2

S I R M.I.C.E Etest M.I.C.E Etest M.I.C.E Etest M.I.C.E Etest M.I.C.E Etest

Amoxicillina ₇ ₇ ₁₀ ₁₂ _{227 (91)} _{225 (90)} ₄ ₄ ₂ ₂

Amoxicillin-clavulanate 154 26 70 1 2 5 10 219 (88) 224 (90) 20 11 4 3

Ampicillin 92 15 143 3 6 10 11 221 (89) 226 (90) 13 3 4 5

Ciprofloxacin 269 8 48 2 1 12 4 257 (76) 281 (85) 49 35 5 4

Cefotaxime 224 1 25 4 6 10 9 225 (90) 221 (88) 9 13 2 1

Gentamicin 275 6 31 3 4 18 17 292 (89) 297 (92) 1 1 3 2

Imipenem 59 1 15 0 0 0 1 70 (93) 61 (81) 5 13 0 0

Levofloxacin 227 6 17 0 0 1 1 209 (84) 192 (77) 36 51 5 6

Total 1,638 65 372 20 26 66 65 1,720 (87) 1,727 (87) 137 131 25 23

a

No resistance breakpoints were found for the strains tested.

TABLE 7Combined error rates for 325 strains of enterobacterial,Pseudomonas, andAcinetobacterspecies for M.I.C.E and Etest compared to broth microdilution

Antimicrobial agent

No. (%) of errors

Minor Major Very major

M.I.C.E Etest M.I.C.E Etest M.I.C.E Etest

Ampicillin 17 (7) 16 (6) 1 (0.4) 1 (0.4) 7 (3) 9 (4)

Amoxicillin-clavulanate 21 (8) 21 (8) 0 0 2 (1) 4 (2)

Cefotaxime 3 (1) 2 (1) 1 (0.4) 1 (0.4) 0 0

Ciprofloxacin 2 (1) 6 (2) 0 0 0 0

Gentamicin 9 (3) 3 (1) 1 (0.3) 1 (0.3) 0 0

Imipenem 1 (1) 4 (5) 0 0 0 0

Levofloxacin 2 (1) 1 (0.4) 0 0 0 0

Total 55 (3) 53 (3) 3 (0.1) 3 (0.1) 9 (0.5) 13 (0.9)

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MRSA, but our observations support the validity of the gradient

endpoint methods to find these isolates.

In more than 90% of minor and major errors, when we

exam-ined the results, the M.I.C. Evaluator strip and Etest MICS were

higher than the broth microdilution results. For enterococci, there

was only one M.I.C.E result where the broth microdilution result

was susceptible. These test systems measure different interactions

between an antimicrobial agent and microorganism. It is to be

expected that there will be some minor differences between the

systems. It is quite satisfying that both gradient endpoint systems

provide MIC measurements that are very close to those of the

broth microdilution reference method.

We expect that these results will establish a mechanism to test

additional M.I.C. Evaluator strip agents against Gram-positive

and Gram-negative bacteria as they become available. A

compan-ion manuscript (11) will report on the validity of the M.I.C.

Eval-uator strips against anaerobes and other fastidious bacterial

spe-cies. All of these studies have shown that the M.I.C. Evaluator

strips provide accurate estimations of the MIC of these bacteria

that are equivalent to results from Etest. Differences with

refer-ence broth microdilution observations are due to the different

formats for these tests and to the mechanics of the interactions

between bacterial cells on agar versus those in broth. For the most

part, the results are very consistent and provide a rationale for the

use of the M.I.C. Evaluator strips for routine MIC antimicrobial

susceptibility testing.

ACKNOWLEDGMENT

This study was supported by an unrestricted grant in aid from Thermo Fisher Scientific, Basingstoke, United Kingdom.

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