Research letters
combination therapy would be recommended to prevent the
emergence of colistin resistance by mutant selection.
The emergence of resistance after exposure to antimicrobial
agents may be due to the selection of antimicrobial-resistant sub-
populations or the occurrence of new mutants through antibiotic
stress. Mutations in two-component systems and LpxACD cause
colistin resistance due to the modification of lipopolysaccharides
in Gram-negative bacteria.8,9 Among 40 A. baumannii isolates,
an amino acid alteration was identified only in a single-step
mutant of one isolate, which showed a P233S substitution in
PmrB in C072 (Table 1), as has been reported in a previous
study.10 No LpxACD mutations were found in this study. In P. aeru-
ginosa, amino acid alterations of PmrAB, PhoPQ and ParRS were
observed in 12 single-step mutants (Table 1). Double amino acid
substitutions were observed in the single-step mutants of four
P. aeruginosa isolates (P70, P83, P88 and P185). V281I in PmrB
and G361R in ParS were each present in two mutants. In
K. pneumoniae, three kinds of amino acid substitution were
observed: S174N in PhoQ and T157P and S208N in PmrB. A T157P
substitution was identified in the single-step mutants of two
K. pneumoniae isolates, 507 BTB and YDJ. Unlike A. baumannii
and P. aeruginosa, amino acid deletions were identified in pmrB
of two single-step mutants of K. pneumoniae. In the single-step
mutant of 08-u-899, amino acid substitution and deletion were
both identified.
Our results indicate that amino acid alterations of PmrAB,
PhoPQ and ParRS occurred in vitro within the period of selection
of single-step mutants. This suggests that colistin treatment can
provoke genetic mutations related to resistance as a mutagen
within a short period in addition to the selection of resistant subpo-
pulations. In short, colistin resistance may occur very easily during
drug use.
In summary, we identified high MPCs of colistin for imipenem-
susceptible and imipenem-resistant A. baumannii, P. aeruginosa
and K. pneumoniae clinical isolates. In single-step mutants,
several amino acid substitutions of two-component regulatory
systems, PmrAB, PhoPQ or ParRS, were also identified. These find-
ings suggest the possibility of the rapid emergence and spread of
colistin resistance by a single mutation. Thus, combination
therapy for colistin treatment of non-fermenter and Enterobacter-
iaceae infections would be necessary to prevent or slow the emer-
gence of colistin resistance.
Acknowledgements
Some A. baumannii, P. aeruginosa and K. pneumoniae isolates used in this
study were obtained from the Asian Bacterial Bank (ABB) of the Asia
Pacific Foundation for Infectious Diseases (APFID), Seoul, Korea.
Funding
This research was supported by the Basic Science Program through the Na-
tional Research Foundation of Korea (NRF) funded by the Ministry of Educa-
tion, Science and Technology (2010-0004848).
Transparency declarations
None to declare.
JAC
References
1 Livermore DM. Current epidemiology and growing resistance of
Gram-negative pathogens. Korean J Intern Med 2012; 27: 128–42.
2 Blondeau JM. New concepts in antimicrobial susceptibility testing: the
mutant prevention concentration and mutant selection window approach.
Vet Dermatol 2009; 20: 383–96.
3 Peck KR, Kim MJ, Cho JY et al. In vitro time– kill studies of antimicrobial
agents against blood isolates of imipenem-resistant Acinetobacter
baumannii, including colistin- or tigecycline-resistant isolates. J Med
Microbiol 2012; 61: 353–60.
Downloaded from https://academic.oup.com/jac/article-abstract/69/1/277/850005 by guest on 21 June 2019
4 Clinical and Laboratory Standards Institute. Performance Standards for
Antimicrobial Susceptibility Testing: Twenty-first Informational Supplement
M100-S21. CLSI, Wayne, PA, USA, 2011.
5 Credito K, Kosowska-Shick K, Appelbaum PC. Mutant prevention
concentrations of four carbapenems against gram-negative rods. Antimicrob
Agents Chemother 2010; 54: 2692–5.
6 Cai Y, Li R, Liang B et al. In vitro antimicrobial activity and mutant
prevention concentration of colistin against Acinetobacter baumannii.
Antimicrob Agents Chemother 2010; 54: 3998– 9.
7 Hawley JS, Murray CK, Jorgensen JH. Colistin heteroresistance in
Acinetobacter and its association with previous colistin therapy. Antimicrob
Agents Chemother 2008; 52: 351–2.
8 Park YK, Choi JY, Shin D et al. Correlation between overexpression and
amino acid substitution of the PmrAB locus and colistin resistance in
Acinetobacter baumannii. Int J Antimicrob Agents 2011; 37: 525– 30.
9 Moffatt JH, Harper M, Harrison P et al. Colistin resistance in Acinetobacter
baumannii is mediated by complete loss of lipopolysaccharide production.
Antimicrob Agents Chemother 2010; 54: 4971– 7.
10 Adams MD, Nickel GC, Bajaksouzian S et al. Resistance to colistin in
Acinetobacter baumannii associated with mutations in the PmrAB
two-component system. Antimicrob Agents Chemother 2009; 53:
3628 – 34.
J Antimicrob Chemother 2014
doi:10.1093/jac/dkt309
Advance Access publication 8 August 2013
Quality control ranges for tylosin 30 mg
and 15 mg discs applicable to
Staphylococcus aureus ATCCw 25923
Monika Buß1,2†, Andrea T. Feßler1†, John Turnidge3,
Thomas Peters2 and Stefan Schwarz1*
1
Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI),
Neustadt-Mariensee, Germany; 2Milchtierherden- Betreuungs- und
Forschungsgesellschaft mbH (MBFG), Wunstorf, Germany;
3
University of Adelaide and SA Pathology at Women’s and Children’s
Hospital, Adelaide, South Australia, Australia
*Corresponding author. Tel: +49-5034-871-241; Fax: +49-5034-871-143;
E-mail: stefan.schwarz@fli.bund.de
†These authors contributed equally to this study.
Keywords: macrolides, disc diffusion, susceptibility testing
277
Research letters

Sir,
Tylosin is a 16-membered macrolide that has never been used in
human medicine. In veterinary medicine, it is currently approved
for therapy of bovine mastitis, pneumonia and arthritis in calves,
bronchitis and bronchopneumonia in dogs, pneumonia, ileitis
and erysipelas in pigs, infectious sinusitis in turkeys and chronic
respiratory disease and necrotizing enteritis in chickens.1
When bacteria are tested for their susceptibility to tylosin,
approved ranges for quality control (QC) strains are indispensable.
QC strains represent an internal control for the test system. The
document VET01-A3 (formerly M31-A3) from the CLSI, the de
for 30 and 15 mg tylosin discs that are applicable to S. aureus ATCCw
25923.
For this, we followed the procedure described in detail in the
CLSI document VET02-A3.5 A total of eight laboratories in
Germany participated in this study, including three federal re-
search laboratories, three county veterinary service laboratories,
one university diagnostic laboratory and one private veterinary
service laboratory. Each laboratory tested two lots of the 30 mg
tylosin disc (one from Biolab, Budapest, Hungary and the other
from MAST Diagnostica, Rheinfeld, Germany) and one lot of the
15 mg tylosin disc (Biolab). Moreover, three different lots of plain

Downloaded from https://academic.oup.com/jac/article-abstract/69/1/277/850005 by guest on 21 June 2019

facto international reference standard, contains tylosin-specific ac-
ceptable QC ranges of MICs for broth microdilution that are applic-
able to Staphylococcus aureus ATCCw 29213 and Enterococcus
faecalis ATCCw 29212.2,3 In addition, tylosin-specific acceptable QC
ranges for antimicrobial disc susceptibility test zone diameters
that are applicable to S. aureus ATCCw 25923 and Streptococcus
pneumoniae ATCCw 49619 have been developed and are included
in the CLSI document VET01-A3.3,4 These zone diameters,
however, refer exclusively to 60 mg discs. Unfortunately, discs that
contain 60 mg of tylosin are hardly commercially available and
routine diagnostic laboratories use the more widely available
30 mg tylosin discs, although no QC criteria have been established
to ensure their correct performance in routine susceptibility
testing. Moreover, the CLSI document VET02-A3 (formerly
M37-A3) states with regard to the disc content that ‘in most
cases, the content of the antimicrobial disc will be the same as
that for other structurally related antimicrobials’.5 The content for
commercially available discs of other macrolides, for which QC
ranges have been approved by the CLSI, are 15 mg for erythromycin
and tilmicosin discs and 30 mg for tulathromycin discs.3 Because of
this anomaly, the aim of the present study was to develop QC ranges
Mueller –Hinton agar (MHA) were purchased as powder from
three different manufacturers (Oxoid, Wesel, Germany; Roth, Karls-
ruhe, Germany; and MAST Diagnostica) and aliquots of them were
distributed to the eight laboratories for use. The agar plates were
prepared in the participating laboratories according to the manu-
facturers’ instructions. Each laboratory tested S. aureus ATCCw
25923 on each MHA lot and each disc on 10 separate days. For com-
parative reasons, all laboratories also tested one lot of the 15 mg
erythromycin disc (Oxoid) on one lot of MHA (Roth) as recom-
mended in the CLSI document VET02-A3.5 This resulted in 80
data points for each individual MHA and tylosin disc lot and a
total of 480 data points for the 30 mg tylosin discs, 240 data
points for the 15 mg tylosin disc and 80 data points for the 15 mg
erythromycin disc. The zone diameters were measured (mm),
rounded to the next integer values and then tabulated and com-
pared between laboratories and reagent lots (MHA and discs
from different manufacturers) as previously described (Tables 1
and 2).4 QC ranges were calculated according to the method of
Turnidge and Bordash,6 the so-called RangeFinder statistical
method.

Table 1. Overall distribution of the zone diameters of S. aureus ATCCw 25923 when tested by using different media lots and disc lotsa

30 mg tylosin disc 15 mg tylosin disc

Zone diameter
(mm) MHA lot A MHA lot B MHA lot C disc lot A disc lot B MHA lot A MHA lot B MHA lot C disc lot A

14
15 1 1
16 1 1
17 12 2 3 17
18 3 1 1 3 7 9 7 23
19 7 3 3 2 11 25 9 11 45
20 30 19 21 15 55 21 23 25 69
21 45 31 33 45 64 11 17 23 51
22 40 29 30 49 50 3 10 8 21
23 19 22 30 41 30 4 2 6
24 10 17 28 39 16 3 3
25 4 24 14 33 9 2 2
26 2 9 1 11 1 1 1
27 5 4 1
28

Total 160 160 160 240 240 80 80 80 240

Mean+SD 21.51+1.51 22.67+2.03 22.30+1.61 22.82+1.77 21.50+1.57 19.23+1.39 20.55+1.83 20.08+1.44 19.95+1.65

a
The proposed QC ranges are indicated with grey shading.

278
Research letters JAC
Table 2. Inter-laboratory comparison of the zone diameters measured when testing S. aureus ATCCw 25923 and 30 and 15 mg tylosin discsa

Laboratory codes (A– H), 30 mg tylosin disc Laboratory codes (A– H), 15 mg tylosin disc

Zone diameter (mm) A B C D E F G H total A B C D E F G H total

14
15 1 1
16 1 1
17 8 8 1 17
18 1 3 4 3 6 1 6 6 1 23

Downloaded from https://academic.oup.com/jac/article-abstract/69/1/277/850005 by guest on 21 June 2019

19 1 8 2 2 13 4 9 9 1 3 1 14 4 45
20 6 20 2 13 3 16 10 70 4 6 10 2 11 11 8 17 69
21 13 12 16 12 7 8 28 13 109 8 1 9 7 9 11 6 51
22 9 14 22 6 14 13 11 10 99 7 1 1 4 6 2 21
23 7 3 13 12 11 16 3 6 71 2 1 2 1 6
24 14 6 6 14 9 6 55 1 1 1 3
25 6 1 6 6 10 13 42 2 2
26 1 1 5 3 2 12 1 1
27 2 2 1 5
28
Total 60 60 60 60 60 60 60 60 480 30 30 30 30 30 30 30 30 240
Meanb 22.6 20.6 22.1 22.2 23.1 23.2 21.0 22.5 22.2 20.6 18.5 20.0 19.8 20.8 20.8 18.9 20.1 20.0
Median 22.5 20 22 22 23 23 21 22 22 21 19 20 19.5 21 21 19 20 20

a
The proposed QC ranges are indicated with grey shading.
b
The mean values were rounded to one decimal place.

All zone diameter results of all participating laboratories obtained the forthcoming CLSI document VET01-S3. Thus, laboratories
with the 15 mg erythromycin disc were within the acceptable range should test the commercially widely available 30 mg tylosin disc
of 22–30 mm. No significant differences could be detected between as the preferred disc.
the three different media lots and the two different 30 mg tylosin
disc lots. Table 1 shows the zone diameters of S. aureus ATCCw
25923 with regard to the different MHA lots and the 30 and 15 mg
tylosin disc lots. The mean diameters of the two lots of the 30 mg
tylosin disc were 22.82 mm (+1.77 mm SD) and 21.50 mm Acknowledgements
(+1.57 mm SD), respectively. The mean diameter value for the We wish to thank all the participants of this study, namely Christin Freitag/
15 mg disc was 19.95 mm (+1.65 mm SD). Table 2 shows the inter- Melanie Hassel (Landesuntersuchungsamt Rheinland-Pfalz, Koblenz,
laboratory comparisons of the zone diameter results obtained for Germany), Rüdiger Hauck (Free University of Berlin, Institute of Poultry
the 30 and 15 mg tylosin discs. Using the method of Turnidge and Diseases, Berlin, Germany), Heike Kaspar (Federal Office of Consumer
Bordash, the proposed QC ranges are 18–26 mm for the 30 mg Protection and Food Safety, Berlin, Germany), Peter Kämpf (Bavarian
Health and Food Safety Authority, Oberschleißheim, Germany), Karin
tylosin disc and 16–24 mm for the 15 mg tylosin disc. As shown in
Knappstein (Max-Rubner-Institut, Kiel, Germany) and Christiane
Figure S1 (available as Supplementary data at JAC Online), Table 1
Werckenthin (Lower Saxony State Office for Consumer Protection and
and Table 2, these proposed QC ranges include 475/480 (99.0%) Food Safety, Food and Veterinary Institute Oldenburg, Germany) for their
of the observed zone diameter values for the 30 mg tylosin discs contribution and excellent cooperation.
and 236/240 (98.3%) of the corresponding zone diameter values
for the 15 mg tylosin disc.
These QC ranges will expand the currently available set of
QC ranges for tylosin and will help the routine diagnostic labora-
tories to validate their results when using tylosin discs that Funding
contain 30 or 15 mg of the antimicrobial agent. Moreover, these This study was financially supported by internal funding of the Institute of
QC ranges will contribute to a harmonization and standardization Farm Animal Genetics of the Friedrich-Loeffler-Institut (FLI).
of tylosin susceptibility testing. Although QC ranges for both
tylosin disc strengths have been determined, those for the
30 mg tylosin disc have been approved by the Veterinary Anti-
microbial Susceptibility Testing subcommittee of the CLSI
during its meeting on 21 June 2013. The QC ranges for the Transparency declarations
30 mg tylosin disc will replace those for the 60 mg tylosin disc in None to declare.

279
Research letters
Supplementary data
Figure S1 is available as Supplementary data at JAC Online (http://jac.
oxfordjournals.org/).
References
1 Veterinary Medicinal Information Service for Drug Application, Toxicology
and Drug Regulation. www.vetidata.de (17 May 2013, date last
accessed).
2 Odland BA, Erwin ME, Jones RN. Quality control guidelines for disk
diffusion and broth microdilution antimicrobial susceptibility tests
with seven drugs for veterinary applications. J Clin Microbiol 2000; 38:
453–5.
3 Clinical and Laboratory Standards Institute. Performance Standards for
Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated
From Animals—Third Edition: Approved Standard VET01-A3 (formerly
M31-A3). CLSI, Wayne, PA, USA, 2008.
4 Anderegg TA, Jones RN, Hall G et al. Initial disk diffusion quality control
guidelines for tylosin tested against Staphylococcus aureus ATCC 25923
and Streptococcus pneumoniae ATCC 49619. Int J Antimicrob Agents
2003; 21: 594–6.
5 Clinical and Laboratory Standards Institute. Development of In Vitro
Susceptibility Testing Criteria and Quality Control Parameters for Veterinary
Antimicrobial Agents—Third Edition: Approved Guideline VET02-A3
(formerly M37-A3). CLSI, Wayne, PA, USA, 2008.
6 Turnidge J, Bordash G. Statistical methods for establishing quality control
ranges for antibacterial agents in Clinical and Laboratory Standards
Institute susceptibility testing. Antimicrob Agents Chemother 2007; 51:
2483– 8.
J Antimicrob Chemother 2014
doi:10.1093/jac/dkt317
Advance Access publication 7 August 2013
Resurgence of penicillin-susceptible
Staphylococcus aureus at a hospital in
New York State, USA
John K. Crane1,2*
1
Division of Infectious Diseases, Room 317 Biomedical Research
Bldg, 3435 Main St., University at Buffalo, Buffalo, NY 14214, USA;
2
Erie County Medical Center, Buffalo, NY, USA
*Corresponding author. Tel: +1-716-829-2676; Fax: +1-716-829-3889;
E-mail: jcrane@buffalo.edu
Keywords: hospital epidemiology, methicillin, antibiotic resistance
Sir,
I read with interest the article by Nissen et al.1 on the treatment of
penicillin-susceptible Staphylococcus aureus (PSSA). Recent arti-
cles on S. aureus have tended to focus on the rising resistance to
antibiotics, including methicillin.2,3 Indeed, methicillin-resistant
S. aureus (MRSA) is now the predominant S. aureus type in most
280
20
susceptible to penicillin
Percentage of strains
15
10
5 ICU isolates
Non-ICU isolates
0
2003 2004 2005 2006 2007 2008 2009 2010 2011 2012
Non-ICU isolates: 2005 2410 1260 1167 1371 1492 1089 998 957
ICU isolates: 207 207 338 327 365 362 318 381 364
Downloaded from https://academic.oup.com/jac/article-abstract/69/1/277/850005 by guest on 21 June 2019
Year
Figure 1. Susceptibility of S. aureus to penicillin G in Erie County Medical
Center, Buffalo, NY, USA. The graph shows the increase in the percentage
of S. aureus isolates that are susceptible to penicillin. These data are based
on patient-unique strains of S. aureus. Data show the percentage of all
S. aureus that are susceptible to penicillin, not just the methicillin-
susceptible subset of strains. Numbers shown below the year indicate the
total number of S. aureus isolates from the two sources for each year.
Analysis by the x2 test for trends was significant (P,0.01) for both ICU
and non-ICU isolates. Linear regression analysis was also significant for
both groups.
hospitals in North America, and the rise of community-acquired
MRSA strains means that patients no longer have to be exposed
to the hospital environment to acquire a resistant Staphylococcus.
Coincident with the rise of MRSA, however, our hospital has also
experienced a resurgence of PSSA (Figure 1). In our 417 bed acute
care hospital in upstate New York, PSSA strains have increased to
13% of all S. aureus among patients in ward beds and to 15%
among patients in intensive care units (ICUs). Many of the other
hospitals in our region have stopped testing S. aureus for suscepti-
bility to penicillin because of the assumption that PSSA has become
extinct. The reason for the increase in PSSA strains is unknown, but I
suspect that heavy reliance on vancomycin has created a niche for
these strains. PSSA appears to have crept back in ‘under the radar’
while our attention has been focused on the more resistant strains.
Our clinical experience is that PSSA strains remain quite virulent;
therefore, antibiotic susceptibility should not be mistaken for a lack
of aggressiveness in vivo. We agree with Nissen et al.1 that penicillin
should be considered the drug of choice for PSSA. Clinical microbiol-
ogy laboratories should be encouraged to test S. aureus strains for
susceptibility to penicillin.
Funding
The observations shown here were part of my routine work as Hospital Epi-
demiologist and were not grant supported.
Transparency declarations
None to declare.
References
1 Nissen JL, Skov R, Knudsen JD et al. Effectiveness of penicillin, dicloxacillin
and cefuroxime for penicillin-susceptible Staphylococcus aureus