Evaluation of antibiotic susceptibility test results: how guilty a laboratory could be?

Abstract

Background
The selection of an appropriate antimicrobial is a challenging task for clinicians. The Kirby-Bauer disk diffusion method is one of the most widely practiced antimicrobial susceptibility tests (AST). It is affected by many factors among which are the media used. Mueller-Hinton agar (MHA) is the standard medium recommended in guidelines. However, these guidelines are not strictly adhered to in some developing countries.

Objectives
Validation of AST results on nutrient agar (NA) medium used as a substitute for MHA by some microbiology laboratories in Alexandria, Egypt.

Methods
A total of 149 clinical bacterial isolates and 3 reference strains: Staphylococcus aureus (S. aureus) ATCC® 25923, Escherichia coli (E. coli) ATCC®25922, and Pseudomonas aeruginosa (P. aeruginosa) ATCC®27853 were comparatively challenged to antibiotics employing MHA and NA.

Results
All antibiotics-reference bacterial strain challenges on NA compared to MHA were unacceptable (> 3 out of limit zones in 30 consecutive days). Considering clinical isolates, the frequency of very major, major, and minor errors on NA was highest in the case of P. aeruginosa (8.98%, 4.08%, and 14.7% respectively) followed by S. aureus (7.6%, 6%, and 8.8% respectively). On the other hand, the least frequency of errors was in the case of Enterobacteriaceae (0%, 0.4%, and 3.2% respectively).

Conclusions and recommendations
Using NA in AST resulted in multiple errors and the high discrepancy in results compared to MHA making it unreliable for susceptibility testing. MHA should not be replaced by NA in AST. Following guidelines and QC measures for AST must be neither bypassed nor underestimated.

Full text

R E S E A R C H Open Access
Evaluation of antibiotic susceptibility test
results: how guilty a laboratory could be?
Mohamed S. M. Nassar, Walaa A. Hazzah
*
and Wafaa M. K. Bakr
Abstract
Background: The selection of an appropriate antimicrobial is a challenging task for clinicians. The Kirby-Bauer disk
diffusion method is one of the most widely practiced antimicrobial susceptibility tests (AST). It is affected by many
factors among which are the media used. Mueller-Hinton agar (MHA) is the standard medium recommended
in guidelines. However, these guidelines are not strictly adhered to in some developing countries.
Objectives: Validation of AST results on nutrient agar (NA) medium used as a substitute for MHA by some
microbiology laboratories in Alexandria, Egypt.
Methods: A total of 149 clinical bacterial isolates and 3 reference strains: Staphylococcus aureus (S. aureus)
ATCC® 25923, Escherichia coli (E.coli) ATCC®25922, and Pseudomonas aeruginosa (P. aeruginosa) ATCC®27853
were comparatively challenged to antibiotics employing MHA and NA.
Results: All antibiotics-reference bacterial strain challenges on NA compared to MHA were unacceptable (> 3
out of limit zones in 30 consecutive days). Considering clinical isolates, the frequency of very major, major, and minor
errors on NA was highest in the case of P. aeruginosa (8.98%, 4.08%, and 14.7% respectively) followed by S. aureus (7.6%,
6%, and 8.8% respectively). On the other hand, the least frequency of errors was in the case of Enterobacteriaceae
(0%, 0.4%, and 3.2% respectively).
Conclusions and recommendations: Using NA in AST resulted in multiple errors and the high discrepancy in
results compared to MHA making it unreliable for susceptibility testing. MHA should not be replaced by NA in
AST. Following guidelines and QC measures for AST must be neither bypassed nor underestimated.
Keywords: CLSI, Mueller-Hinton agar, Inhibition zones, Disk diffusion method, Quality control
1 Introduction
Antibiotic resistance has become a serious public health
problem all over the world. Nearly two million people in
the USA acquire nosocomial infections every year,
resulting in 90,000 deaths. More than 70% of the bac-
teria that causes these infections are resistant to at least
one of the antibiotics commonly used in treatment [1].
This makes the selection of an appropriate agent an in-
creasingly more challenging task that has made clini-
cians more dependent on data from in-vitro AST [2].
In the industrial world, the Kirby-Bauer disk diffusion
method is a standard procedure for the susceptibility test-
ing of bacterial isolates. When the test is performed fol-
lowing a standard procedure, it gives reliable results and
can predict clinical efficacy of the antibiotics tested [3,4].
It has been recognized for years that the general adoption
of antimicrobial susceptibility test (AST) method, so stan-
dardized as to minimize the influence of variables, would
be a great advance. The validity of AST depends on rigid
standardization of every feature of the test, including par-
ticularly the composition of the medium used [5]. The
standard medium for the Kirby-Bauer method of suscepti-
bility testing is Mueller-Hinton agar (MHA) [6]. Because
of the number of difficulties and financial issues, MHA is
not a feasible option in many developing countries, and
instead, NA is used for AST [7]. We performed a pilot
study on 30 randomly selected clinical microbiology
laboratories in Alexandria, using a questionnaire sheet to
assess their compliance to Clinical and Laboratory Stan-
dards Institute (CLSI) guidelines for AST by the disk dif-
fusion method. Most of the laboratories did not perform
* Correspondence: walaahazzah@gmail.com;walaa.hazzah@alexu.edu.eg
Department of Microbiology, High Institute of Public Health, Alexandria
University, Alexandria, Egypt
Journal o
f
the Egyptia
n
P
ub
li
c
H
ea
l
t
h A
ssoc
i
at
i
on
© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made.
Nassar et al. Journal of the Egyptian Public Health Association (2019) 94:4
https://doi.org/10.1186/s42506-018-0006-1

internal quality control (QC), and non-adherence to the
type of medium was one of the frequent errors encoun-
tered. This study aimed at validating the AST results
when performed using NA as a substitute for MHA by
some laboratories.
2 Material and methods
This comparative cross-section study was carried out dur-
ing a 9-month period from June 2015 through February
2016 at the Microbiology Laboratory of the High Institute
of Public Health (HIPH), Alexandria, Egypt.
Three reference bacterial strains, namely Staphylococ-
cus aureus (S. aureus) ATCC® 25923, Escherichia coli
(E. coli) ATCC® 25922, and Pseudomonas aeruginosa (P.
aeruginosa) ATCC® 27853, were used for the internal
QC of AST for 30 consecutive days according to the
CLSI guidelines [6]. We performed the internal QC of
AST using Oxoid antibiotic disks with reference strains
on two media (MHA and NA), followed by comparing
the performance of both media in AST of 149 clinical
bacterial isolates (Table 1). All clinical bacterial isolates
were identified by morphological and biochemical tests
according to standard methods [8].
Zone diameters of susceptibility testing results were
categorized as sensitive, intermediate, or resistant based
on the CLSI breakpoint criteria [6]. Susceptibility testing
results on MHA were compared with those of NA.
Agreements and discrepancies in the results of direct
disk diffusion using NA as a medium and MHA were
classified as follows: agreement (identical result on NA
and MH agar), very major error (susceptible on NA but
resistant on MH agar), major error (resistant on NA but
susceptible on MH agar), and minor error (susceptible
or resistant on NA and intermediate on MH agar, or
vice versa) [9,10].
2.1 Statistical analysis
Data were analyzed using SPSS version 21 (IBM Corp,
USA).
3 Results
In our study, 30 laboratories were assessed for their
compliance with CLSI guidelines for AST by the disk
diffusion method. Only three laboratories (10%) were
adequately complying with the guidelines. Most labora-
tories do not perform internal QC or use QC strains,
and antibiotic disk brands were used indifferently ac-
cording to availability and without prior testing. The
type of medium used is one of the frequent events en-
countered (56.7%) causing lack of compliance with the
CLSI guidelines for AST. Instead of MHA, NA and
tryptic soy agar were used by 11 (36.7%) and 6 (20%) la-
boratories respectively (results not shown).
Comparing NA to MHA, internal QC testing of the
three reference bacterial strains over 30 consecutive days
showed that all antibiotics/organism challenges on NA
were unacceptable (Table 2). That was reflected on the
AST results of clinical isolates cultured on both media
(Table 3), where the frequency of very major errors,
major errors, and minor errors of NA compared to MH
was highest in the case of P. aeruginosa (8.98%, 4.08%,
and 14.7% respectively) followed by S. aureus (7.6%, 6%,
and 8.8% respectively). On the other hand, the least fre-
quency of errors was in the case of Enterobacteriaceae
(0%, 0.4%, and 3.2% respectively).
4 Discussion
To evaluate the validity of AST results using NA, QC
strains were used for AST in uniform conditions in-
cluding inoculum density, timing of disk application,
temperature of incubation, incubation time, size of the
plate, depth of the agar medium, proper spacing of the
antibiotic disks, and potency of antibiotic disks. Appro-
priate QC organisms should be tested daily for all anti-
microbial agents routinely included in the antimicrobial
battery until a laboratory achieves “satisfactory per-
formance.”CLSI makes the definition of “satisfactory
performance”as obtaining unacceptable results in no
more than 1 out of 20 or 3 out of 30 results obtained in
consecutive test days for each antimicrobial agent/or-
ganism combination. Once this satisfactory perform-
ance is obtained, a laboratory can convert from daily
QC testing to weekly QC testing. If all QC test results
are within the acceptable limits, the laboratory can con-
tinue weekly testing; however, on occasions when a
modification in the test is made, consecutive QC testing
is required [6].
All antimicrobial agent-reference organism combina-
tions using NA were unacceptable compared to MHA. All
antibiotic-organism combination showed more than three
misreading over 30 successive days; hence, none of the re-
sults would be accepted according to CLSI guidelines.
Muller-Hinton agar is a loose agar that allows for bet-
ter diffusion of the antibiotics than most other media.
A better diffusion leads to a truer zone of inhibition
[11]. This criterion was missing in NA manifested by
smaller IZs with TOB, CN, and P when testing P. aeru-
ginosa ATCC® 27853, AZM and DA when testing S.
Table 1 Clinical bacterial isolates
Clinical bacterial isolates Number
Staphylococcus aureus 50
Pseudomonas aeruginosa 49
Escherichia coli 17
Klebsiella pneumoniae 20
Proteus vulgaris 3
Proteus mirabilis 10
Nassar et al. Journal of the Egyptian Public Health Association (2019) 94:4 Page 2 of 5

aureus ATCC® 25923, and AMP, KZ, TOB, and CN
when testing E. coli ATCC® 25922 (Table 2). Both the
para-aminobenzoic acid (PABA) and thymine/thymi-
dine content in MHA are reduced to a minimum, thus
markedly reducing the inactivation of sulfonamides and
trimethoprim when the medium is used for testing the
susceptibility of bacterial isolates to these antimicrobics
[11]. Thirteen errors with smaller inhibition zone over
30 consecutive days were detected on testing SXT for S.
aureus ATCC® 25923 using NA (Table 2).
The inability of AST to determine a susceptible re-
sult for an organism that is susceptible to the anti-
microbial agent being tested is considered a major
error (false resistant). Conversely, the inability to de-
tect resistance is assessed as “very major error”(false
sensitive). Concerning CLSI guidelines, there is a mini-
mum level of acceptable interpretative errors in sus-
ceptibility testing which are quite restrictive. Very
major errors should not exceed 1.5%, while major er-
rors should not exceed 3.0%, and overall categorical
agreement should equal or exceed 90% for each organ-
ism antibiotic challenge [10,12,13].
In our study, the discrepancies between the suscepti-
bility results obtained by NA and the standard MHA
were obvious when testing clinical isolates with total
errors of 27.76%, 22.4%, and 3.6% with P. aeruginosa,S.
aureus, and Enterobacteriaceae respectively. Also, very
major errors and major errors were beyond the accept-
able level of CLSI guidelines (8.98% and 7.6% very major
errors and 4.08% and 6% major errors for P. aeruginosa
and S. aureus isolates respectively).
Very major error may lead to the initiation of inad-
equate antimicrobial therapy and may have fatal conse-
quences especially in severely ill patients where these
antibiotics are common first-line substances. On the
other hand, major errors deprive the patients of treat-
ment with an effective antibiotic and lead to the use of
second-choice drugs, usually more recent and expensive,
and thus contribute to economic losses and the selection
of resistant strains [12].
Although none of the AST results for E. coli ATCC®
25922 was acceptable when using NA (Table 2), we
found a remarkable unexplained agreement in AST re-
sults of Enterobacteriaceae on both NA and MHA
(96.4%) with no very major error and 0.4% major error
(Table 3). Even if the AST results showed full agree-
ment as in the case of Enterobacteriaceae,theissueof
lacking data on specific breakpoints concerning the use
of NA remains.
Because NA is a general purpose medium rather than
standard susceptibility testing medium, there is hardly any
data comparing it with MHA in susceptibility testing.
Donkor et al. compared NA with MHA in antimicrobial
susceptibility testing of Salmonella Typhi and S. aureus
isolates. They reported that the overall discrepancy in sus-
ceptibility results between NA and MHA was 8.9%, and
this discouraged the use of NA in the Kirby-Bauer method
as practiced by some laboratories, due to the considerable
Table 2 Antibiotic susceptibility test results of reference bacterial strains on MH agar versus NA during 30days of QC testing, Alexandria,
2015–2016
Reference strains Antibiotic tested Out of range results/30 QC days* (compared to CLSI ranges)
MH NA
Larger IZs Smaller IZs Larger IZs Smaller IZs
P. aeruginosa ATCC® 27853 Ceftazidim (CAZ) 0/30 0/30 13/30 2/30
Gentamicin (CN) 0/30 0/30 4/30 13/30
Tobramycin (TOB) 0/30 0/30 0/30 15/30
Piperacillin (PRL) 1/30 2/30 6/30 9/30
Ciprofloxacin (CIP) 2/30 0/30 19/30 2/30
E. coli ATCC®25922 Ampicillin (AMP) 0/30 3/30 0/30 13/30
Cefazolin (KZ) 0/30 1/30 0/30 13/30
Gentamicin (CN) 0/30 2/30 0/30 6/30
Tobramycin (TOB) 0/30 1/30 0/30 9/30
Tazobactam-piperacillin (TZP) 0/30 0/30 2/30 6/30
S. aureus ATCC®25923 Azithromycin (AZM) 0/30 2/30 0/30 23/30
Clindamycin (DA) 0/30 2/30 0/30 9/30
Cefoxitine (FOX) 0/30 2/30 6/30 1/30
Penicillin (P) 1/30 2/30 16/30 2/30
Sulfamethoxazol-trimethoprim (SXT) 0/30 0/30 4/30 13/30
*QC performed over 30 consecutive days
Nassar et al. Journal of the Egyptian Public Health Association (2019) 94:4 Page 3 of 5

error margin this medium may introduce into susceptibil-
ity results [14].
Though our results cannot be generalized on the la-
boratories’performanceasitwasapilotstudycon-
ducted on a small number of microbiology laboratories
and focusing on one factor affecting AST, it highlights
the great importance of standardizing every feature in
this test to improve the validity of AST reporting of a
laboratory.
5 Conclusion and recommendations
The high deviation of AST results observed between
MHA and NA raises doubts about the reliability of the
NA for susceptibility testing and should not be used as a
substitute for MHA. Lack of adherence to strict standard-
ized AST seriously affects treatment strategies for the in-
fections by misleading the physician about the sensitivity
and resistance of the isolate to different antibiotics with a
deleterious effect on the patients’outcome. Hence, strict
adherence to the guidelines and QC measures for AST
must be neither bypassed nor underestimated.
Acknowledgements
Not applicable.
Funding
This study is self-funded by the authors.
Availability of data and materials
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
Authors’contributions
WMKB contributed to the conception of the study. MSMN performed the
analysis, and WAH shared the interpretation of data. All authors contributed
to the drafting of the article, the revision of the article for critically important
intellectual content, and the final approval of the version to be published.
Ethics approval and consent to participate
The study did not involve human beings or human samples. We performed
a pilot study on 30 randomly selected clinical microbiology laboratories in
Alexandria to assess their compliance to guidelines. A verbal consent was
sought from participating laboratories. according to the laboratories directors’
request, and we only showed the most important results and kept all their data
anonymous as agreed upon. The ethics committee of the High Institute
of Public health approved the study.
Consent for publication
Not applicable.
Table 3 Antibiotic susceptibility test results of clinical bacterial isolates on MHA versus NA using CLSI zone size interpretative table
HIPH, Alexandria, 2015–16
Antibiotic/clinical isolate SIR agreement* Very major error Major error Minor error
No. (%) No. (%) No. (%) No. (%)
P. aeruginosa (n= 49)
Ceftazidim (CAZ) 46 (93.90) 0 (0.00) 3 (6.10) 0 (0.00)
Gentamicin (CN) 38 (77.55) 8 (16.30) 2 (4.10) 1 (2.00)
Tobramycin (TOB) 33 (67.30) 10 (20.40) 2 (4.10) 4 (8.20)
Piperacillin (PRL) 24 (49.00) 0 (0.00) 1 (2.00) 24 (49.00)
Ciprofloxacin (CIP) 36 (73.50) 4 (8.20) 2 (4.10) 7 (14.30)
Total 177 (72.24) 22 (8.98) 10 (4.08) 36 (14.70)
Enterobacteriaceae (n= 50)
Ampicillin (AMP) 50 (100.00) 0 (0.00) 0 (0.00) 0 (0.00)
Cefazolin (KZ) 46 (92.00) 0 (0.00) 1 (2.00) 3 (6.00)
Gentamicin (CN) 50 (100.00) 0 (0.00) 0 (0.00) 0 (0.00)
Tobramycin (TOB) 47 (94.00) 0 (0.00) 0 (0.00) 3 (6.00)
Tazobactam-piperacillin (TZP) 48 (96.00) 0 (0.00) 0 (0.00) 2 (4.00)
Total 241 (96.40) 0 (0.00) 1 (0.40) 8 (3.20)
S. aureus (n= 50)
Azithromycin (AZM) 43 (86.00) 0 (0.00) 3 (6.00) 4 (8.00)
Clindamycin (DA) 41 (82.00) 1 (2.00) 0 (0.00) 8 (16.00)
Cefoxitine (FOX) 33 (66.00) 10 (20.00) 7 (14.00) 0 (0.00)
Penicillin (P) 44 (88.00) 2 (4.00) 4 (8.00) 0 (0.00)
Sulfamethoxazol-trimethoprim (SXT) 33 (66.00) 6 (12.00) 1 (2.00) 10 (20.00)
Total 194 (77.60) 19 (7.60) 15 (6.00) 22 (8.80)
*Category agreement with respect to susceptible (S), intermediate (I), and resistant (R) test results
Nassar et al. Journal of the Egyptian Public Health Association (2019) 94:4 Page 4 of 5

Competing interests
The authors declare that they have no competing interests.
Publisher’sNote
Springer Nature remains neutral with regard to jurisdictional claims in published
maps and institutional affiliations.
Received: 2 November 2018 Accepted: 20 December 2018
References
1. FDA. US battle of the bugs: fighting antibiotic resistance. [Internet] Updated:
05/04/2016. Available from: http://www.fda.gov/drugs/resourcesforyou/
consumers/ucm143568.htm. Accessed 5 May 2017.
2. Shigemura K, Tanaka K, Adachi M, Yamshita M, Arakawa S, Fujisawa M.
Chronological change of antibiotic use and antibiotic resistance in Escherichia
coli causing urinary tract infections. J Infect Dis Chemother. 2011;17:646–51.
3. King A, Brown DFJ. Quality assurance of antimicrobial susceptibility testing
by disc diffusion. J Antimicrob Chemother. 2001;48:71–6.
4. Clinical and Laboratory Standards Institute (CLSI). Performance standards for
antimicrobial susceptibility testing; 20
th
Informational supplement M100-
S20. Wayne: CLSI; 2010.
5. Garrod LP, Waterworth PM. A study of antibiotic sensitivity testing with
proposals for simple uniform methods. J Clin Pathol. 1971;24:779–89.
6. Clinical and Laboratory Standards Institute (CLSI). Performance standards for
antimicrobial susceptibility testing; 23
rd
Informational supplement M100-
S23. Wayne: CLSI; 2013.
7. Niederstebruch N, Sixt D. Standard nutrient agar 1 as a substitute for blood-
supplemented Müeller-Hinton agar for antibiograms in developing
countries. Eur J Clin Microbiol Infect Dis. 2013;32:237–41.
8. Forbes BA, Sahm DF, Weissfeld AS. Bailey & Scott’s diagnostic microbiology.
12th ed. St. Louis: Mosby Elsevier; 2007.
9. Edelmann A, Pietzcker T, Wellinghausen N. Comparison of direct disk
diffusion and standard microtitre broth dilution susceptibility testing of
blood culture isolates. J Med Microbiol. 2007;56:202–7.
10. Stuckey S. Automated systems: an overview. In: Schwalbe R, Steele-Moore L,
Goodwin AC, editors. Antimicrobial susceptibility testing protocols. Ch 5.
Boca Raton: CRC Press Taylor & Francis; 2007. p. 81–9.
11. Aryal S. Mueller Hinton agar (MHA) –composition, principle, uses and
preparation. Microbiology info. [Internet] August 24, 2015. Updated: September
20, 2015. Available from: http://www.microbiologyinfo.com/mueller-hinton-
agar-mha-composition-principle-uses-and-preparation. Accessed 15 Aug 2016.
12. Jean SS, Liao CH, Sheng WH, Lee WS, Hsueh PR. Comparison of commonly
used antimicrobial susceptibility testing methods for evaluating
susceptibilities of clinical isolates of Enterobacteriaceae and
nonfermentative Gram-negative bacilli to cefoperazone–sulbactam. J
Microbiol Immunol Infect. 2017;50(4):454–3.
13. Clinical and Laboratory Standards Institute (CLSI). Methods for dilution
antimicrobial susceptibility tests for bacteria that grow aerobically, approved
standard M7-A7. Wayne: CLSI; 2007.
14. Donkor ES, Nortey T, Opintan J, Akyeh ML. Antimicrobial susceptibility of
Salmonella Typhi and Staphylococcus aureus isolates and the effect of some
media on susceptibility testing results. Internet J Microbiol. 2007;4:1–6.
Nassar et al. Journal of the Egyptian Public Health Association (2019) 94:4 Page 5 of 5