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Journal of Medical Microbiology (2014), 63, 242–247
DOI 10.1099/jmm.0.061788-0
Antimicrobial susceptibility and mechanisms of
high-level macrolide resistance in clinical isolates of
Moraxella nonliquefaciens
Shotaro Nonaka,1 Kosuke Matsuzaki,13 Tomoya Kazama,1
Hiroyuki Nishiyama,2 Yoko Ida,3 Saho Koyano,4 Kazunari Sonobe,5
Noboru Okamura1 and Ryoichi Saito1
Correspondence
Ryoichi Saito
[email protected]
1
Department of Microbiology and Immunology, Graduate School of Health Care Sciences,
Tokyo Medical and Dental University, Tokyo, Japan
2
Clinical Laboratory Department, Surugadai Nihon University Hospital, Tokyo, Japan
3
Department of Clinical Laboratory, Kyorin University Hospital, Tokyo, Japan
4
Department of Infection Control and Prevention, The University of Tokyo Hospital, Tokyo, Japan
5
Department of Clinical Laboratory, Nippon Medical School, Tokyo, Japan
Received 17 April 2013
Accepted 4 November 2013
We investigated antimicrobial susceptibility and the molecular mechanism involved in conferring
high-level macrolide resistance in 47 clinical isolates of Moraxella nonliquefaciens from Japan.
Antimicrobial susceptibility was determined using Etest and agar dilution methods. Thirty-two
erythromycin-non-susceptible strains were evaluated for the possibility of clonal spreading, using
PFGE. To analyse the mechanism related to macrolide resistance, mutations in the 23S rRNA
gene and the ribosomal proteins, and the presence of methylase genes were investigated by PCR
and sequencing. The efflux system was examined using appropriate inhibitors. Penicillin,
ampicillin, amoxicillin, cefixime, levofloxacin and antimicrobials containing b-lactamase inhibitors
showed strong activity against 47 M. nonliquefaciens isolates. Thirty-two (68.1 %) of the 47
isolates showed high-level MICs to macrolides (MIC ¢128 mg l”1) and shared the A2058T
mutation in the 23S rRNA gene. The geometric mean MIC to macrolides of A2058T-mutated
strains was significantly higher than that of WT strains (P,0.0001). Thirty-two isolates with highlevel macrolide MICs clustered into 30 patterns on the basis of the PFGE dendrogram, indicating
that the macrolide-resistant strains were not clonal. In contrast, no common mutations of the
ribosomal proteins or methylase genes, or overproduction of the efflux system were observed in
A2058T-mutated strains. Moreover, of the 47 M. nonliquefaciens strains, 43 (91.5 %) were bro-1
and 4 (8.5 %) were bro-2 positive. Our results suggest that most M. nonliquefaciens clinical
isolates show high-level macrolide resistance conferred by the A2058T mutation in the 23S rRNA
gene. This study represents the first characterization of M. nonliquefaciens.
INTRODUCTION
Moraxella nonliquefaciens, a Gram-negative coccobacillus,
is part of the normal microbiota of the human respiratory
tract (Vaneechoutte et al., 2012). Previous reports have
suggested that M. nonliquefaciens causes various infectious diseases, such as endocarditis, endophthalmitis and
3Present address: Department of Clinical Laboratory, Tokorozawa City
Citizen’s Medical Center Clinical Inspection, Saitama, Japan.
Abbreviations: TSA, tryptone soya agar; BHI, brain heart infusion; PabN,
phenylalanine arginine b-naphthylamide; CCCP, carbonyl cyanide mchlorophenylhydrazone.
The GenBank/EMBL/DDBJ accession numbers for the partial 23S
gene sequences of M. nonliquefaciens are AB818473–AB818489.
242
pneumonia (Davis et al., 2004; Laukeland et al., 2002;
Rafiq et al., 2011), and it has been reported to show
susceptibility to various antimicrobials, including macrolides (Davis et al., 2004; Laukeland et al., 2002; Vaneechoutte
et al., 2012).
Macrolides act by binding to the 23S rRNA gene and prevent
elongation of the peptide chain (Leclercq & Courvalin,
1998). Currently, clarithromycin and azithromycin are
frequently used for the treatment of respiratory tract
infections (Grover et al., 2012; Higashi & Fukuhara, 2009;
Kawai et al., 2012). Mechanisms of resistance against these
antimicrobials, including in Moraxella catarrhalis, which is
among the Moraxella species most frequently isolated from
clinical samples, are known to involve active efflux of
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Macrolide resistance in M. nonliquefaciens
antimicrobials and modification of the ribosomal target by
enzymes such as rRNA methylase, or mutations in domain V
of the 23S rRNA gene (Chironna et al., 2011; Chisholm et al.,
2010; Li et al., 2011; Saito et al., 2012; Tait-Kamradt et al.,
2000; Versalovic et al., 1997). Another mechanism, involving
mutations in the ribosomal proteins L4 and L22 encoded by
rplD and rplV, respectively, has been reported to underlie
resistance in some clinical isolates (Berisio et al., 2006; TaitKamradt et al., 2000).
There is, however, a need for more data from clinical
isolates to assess the risk of emergence of antimicrobialresistant M. nonliquefaciens strains. Therefore, we investigated the antimicrobial susceptibility of 47 clinical isolates
of M. nonliquefaciens and characterized the molecular
mechanism related to high-level resistance to macrolides in
these strains. We demonstrated for the first time that a
point mutation in the 23S rRNA gene confers high-level
resistance to macrolides in M. nonliquefaciens.
METHODS
Bacterial strains and culture conditions. A total of 47 M.
nonliquefaciens clinical isolates collected at four university hospitals in
Tokyo, Japan, from October 2008 to July 2009, and a type strain, M.
nonliquefaciens ATCC 17953, were examined in this study. The
clinical isolates were obtained from nasopharyngeal specimens of
patients with respiratory tract infections. They were identified using
the VITEK 2 system (SYSMEX; bioMérieux) and 16S rRNA gene
sequencing using primers 10F and 800R (Table 1). M. nonliquefaciens
strains were cultured at 37 uC on tryptone soya agar (TSA) plates
containing 5 % sheep blood (Nippon Becton Dickinson) and brain
heart infusion (BHI) agar plates (Oxoid) in an atmosphere of 95 % air
and 5 % CO2. Cultures were maintained in 10 % skimmed milk and
stored at 280 uC until required for subsequent experiments.
Antimicrobial susceptibility testing. The antimicrobial susceptibil-
ity of M. nonliquefaciens strains was determined using the Etest method
(AB BIODISK) on TSA containing 5 % sheep blood, according to the
manufacturer’s recommendations. The following antibiotics were tested
by Etest: penicillin, ampicillin, amoxicillin, ampicillin-sulbactam,
amoxicillin-clavulanic acid, cefixime and levofloxacin. Antimicrobial
susceptibility to erythromycin (Wako Pure Chemical), clarithromycin
(Taisho Pharmaceutical), azithromycin (Tokyo Chemical Industry) and
josamycin (Astellas Pharma) was determined by the agar dilution
method, using BHI agar. MICs of antimicrobials were interpreted
according to the Clinical and Laboratory Standards Institute (CLSI)
criteria for M. catarrhalis (CLSI, 2010). Staphylococcus aureus ATCC
29213 was used for quality control.
PFGE. Genomic DNA of 32 erythromycin-non-susceptible M.
nonliquefaciens strains was prepared in agarose plugs treated with
proteinase K (Takara Bio). The DNA was digested with 25 U SpeI (New
England Biolabs) and electrophoresis was performed using a CHEF
DRII system (Bio-Rad). Fingerprinting II software (Bio-Rad) was used
to analyse the DNA restriction patterns and determine their similarity,
based on calculation of the Dice similarity coefficient and using the
unweighted pair group method with arithmetic mean algorithm.
DNA sequencing of macrolide-resistance targets and detection
of methylase and bro b-lactamase genes. Bacterial genomic DNA
was extracted by boiling. The 23S rRNA gene, rplD and rplV were
amplified by PCR using the relevant primer sets (Table 1), as follows:
Table 1. Primers used in this study
Target gene/primer
16S rRNA
16S-10F
16S-800R
23S rRNA
23S-1930F
23S-2488R
rplD
L4-24F
L4-589R
rplV
L22-32F
L22-325R
erm(A)
ermA-F
ermA-R
erm(B)
ermB-F
ermB-R
erm(C)
ermC-F
ermC-R
erm(F)
ermF-L
ermF-R
http://jmm.sgmjournals.org
Sequence (5’A3’)
Reference
GTTTGATCCTGGCTCA
TACCAGGGTATCTAATCC
Saito et al. (2012)
GGTAGCGAAATTCCTTGTCG
CGCCGTCGATATGAACTCTT
Saito et al. (2012)
This study
TGCAGCGGTTGAACTATCAG
CCTCAAATTGTTTGGCTGCT
Saito et al. (2012)
CCATTTCGGCACAGAAAGTAA
CCCCTACTTTAACAGTGATGTGA
Saito et al. (2012)
AGCGGTAAACCCCTCTGAG
TAGTGACATTTGCATGCTTCAA
Nakaminami et al. (2008)
AGTAACGGTACTTAAATTGTTTAC
GAAAAGGTACTCAACCAAATA
Sutcliffe et al. (1996)
GCTAATATTGTTTAAATCGTCAAT
TCAAAACATAATATAGATAAA
Sutcliffe et al. (1996)
CGGGTCAGCACTTTACTATTG
GGACCTACCTCATAGACAAG
Chung et al. (1999)
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243
S. Nonaka and others
¡0.016 mg l21; cefixime, ¡0.016 mg l21; levofloxacin,
0.064 mg l21; erythromycin, ¡0.125 mg l21; clarithromycin, ¡0.125 mg l21; azithromycin, ¡0.125 mg l21;
josamycin, 1 mg l21.
3 min of initial denaturation at 95uC, 30 cycles each consisting of 30 s
at 95 uC, 30 s at 53 uC and 1 min at 72 uC and 3 min of final
extension at 72uC. PCR amplicons were sequenced on an ABI PRISM
3100 genetic analyser (Applied Biosystems). DNA sequences were
compared with the M. nonliquefaciens ATCC 17953 sequence
(GenBank accession no. AB745464 for 23S rRNA, AB745465 for
rplD and AB745466 for rplV) using BLAST.
The MIC90 values of antibiotics, excluding macrolides, for
the isolates in this study were: penicillin, 4 mg l21;
ampicillin, 0.25 mg l21; amoxicillin, 0.5 mg l21; cefixime,
0.25 mg l21; levofloxacin, 0.064 mg l21. The MIC90 values
of ampicillin and amoxicillin containing a b-lactamaseinhibitor were 0.125 and 0.125 mg l21, respectively. These
antibiotics showed strong activity against M. nonliquefaciens.
The presence of the methylase genes erm(A), erm(B), erm(C) and
erm(F) was screened for using the relevant primer sets (Table 1) as
described previously (Chung et al., 1999; Nakaminami et al., 2008;
Roberts et al., 1999; Saito et al., 2012; Sutcliffe et al., 1996). S. aureus,
Streptococcus pneumoniae and Bacteroides vulgatus were used as positive
controls for erm(A) and erm(C), erm(B) and erm(F), respectively.
In contrast, macrolides were less active against M.
nonliquefaciens than the other seven tested antibiotics: the
MIC90 values of erythromycin, clarithromycin, azithromycin and josamycin were .512, 512, 512 and 256 mg l21,
respectively. Based on the CLSI breakpoints for M.
catarrhalis, the non-susceptible rates of amoxicillin–
clavulanic acid (.4 mg l21), levofloxacin (.2 mg l21),
erythromycin (.2 mg l21), clarithromycin (.1 mg l21)
and azithromycin (.0.125 mg l21) were 0 % (0/47), 0 %
(0/47), 68.1 % (32/47), 68.1 % (32/47), and 68.1 % (32/47),
respectively.
PCR amplification of bro was performed as described previously
(Khan et al., 2010).
Analysis of the efflux system. To investigate the role of efflux
systems in resistance to macrolides, efflux pump activity was assessed
by the addition of two different inhibitors, phenylalanine arginine
b-naphthylamide (PAbN; Sigma-Aldrich) and carbonyl cyanide
m-chlorophenylhydrazone (CCCP; Nacalai Tesque) to the growth
medium at the final concentration of 10 and 0.1 mg l21, respectively.
A greater than twofold decrease in MICs with and without inhibitors
for each antibiotic was considered to indicate a significant change.
The effects of the relevant inhibitors were determined at least twice.
Statistical analysis. A Mann–Whitney U test was used to determine
statistical differences in MICs. In all analyses, P,0.05 was considered
significant.
PFGE
To investigate the possibility of clonal spreading of
macrolide-resistant M. nonliquefaciens strains, 32 erythromycin-non-susceptible strains were evaluated by PFGE.
The resulting dendrogram showed 30 PFGE patterns (Fig.
1). Strains in which a similar band pattern was detected
(Mn8 and Mn10, as well as Mn13 and Mn19) had not been
isolated from the same hospital.
RESULTS
Antimicrobial susceptibility testing
The results of antimicrobial susceptibility testing of 47
clinical isolates to 12 antibiotics are shown in Table 2. The
MIC of antibiotics for an M. liquefaciens type strain ATCC
17953 were as follows: penicillin, 0.040 mg l21; ampicillin,
¡0.016 mg l21; amoxicillin, ¡0.016 mg l21; ampicillin–
sulbactam, ¡0.016 mg l21; amoxicillin–clavulanic acid,
Analysis of macrolide-resistance targets
In comparison with the 23S rRNA gene sequence (nt 1964–
2430; Eschericia coli numbering, GenBank accession no.
Table 2. Antimicrobial susceptibility in 47 clinical isolates of M. nonliquefaciens
MIC (mg l”1)
Antimicrobial
¡0.125 0.25
Penicillin
Ampicillin
AMP/SUL
Amoxicillin
AMC
Cefixime
Levofloxacin
Erythromycin
Clarithromycin
Azithromycin
Josamycin
36
47
7
45
26
47
7
9
15
0.5
1
2
4
8
1
10
6
1
10
16
13
1
20
2
18
16
4
3
1
4
5
1
1
2
16
32
64
128
256
1
6
6
12
19
17
MIC90 (mg l”1)
2
0.125
0.064
0.25
0.064
0.125
0.064
512
256
128
128
4
0.25
0.125
0.5
0.125
0.25
0.064
.512
512
512
256
512 .512
3
1
7
15
MIC50 (mg l”1)
17
17
4
15
2
1
AMP/SUL, ampicillin–sulbactam; AMC, amoxicillin–clavulanic acid.
244
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Journal of Medical Microbiology 63
100
80
60
40
Macrolide resistance in M. nonliquefaciens
.Mn13
.Mn19
.Mn43
.Mn37
.Mn38
.Mn16
.Mn25
.Mn27
.Mn8
.Mn10
nt 2058 was significantly lower than those of 32 erythromycin-non-susceptible strains bearing the A2058T mutation (P,0.0001; Table 3).
Furthermore, five different amino acid sequence patterns
in L4 and two in L22 were found among the 47 M.
nonliquefaciens (data not shown). No sequence patterns
present only in erythromycin-nonsusceptible strains were
observed.
To investigate the presence of methylase genes, a PCR
detection assay was performed. We did not detect erm(A),
erm(B), erm(C) or erm(F) in any of the 47 clinical isolates.
.Mn23
.Mn6
.Mn45
.Mn52
.Mn22
.Mn24
.Mn2
Analysis of bro b-lactamase genes
Among the 47 M. nonliquefaciens clinical isolates, 43
(91.5 %) were positive for bro-1 and 4 (8.5 %) for bro-2.
The results of DNA sequencing of bro-1 (165 bp) and bro-2
(144 bp) showed 100 % similarity to those of M. catarrhalis
(GenBank accession nos Z54180 and Z54181, respectively).
.Mn40
.Mn12
.Mn1
.Mn46
.Mn63
.Mn36
.Mn39
.Mn4
.Mn17
.Mn26
.Mn18
.Mn21
.Mn49
.Mn47
Analysis of the efflux system
MICs of erythromycin for the 47 M. nonliquefaciens isolates
were evaluated in the absence or presence of efflux pump
inhibitors. In the presence of PAbN, the MICs of 61.7 %
(29/47) isolates remained unchanged, 25.5 % (12/47) were
decreased twofold and 8.5 % (4/47) were decreased fourfold (three erythromycin-non-susceptible strains: ¢512 to
256 mg l21; one erythromycin-susceptible strain: 0.5 to
¡0.125 mg l21) compared with the MICs in the absence of
PAbN; 6.4 % (3/47) did not grow. On the other hand, in
the presence of CCCP, the MICs of 95.7 % (45/47)
remained similar to those observed in the absence of
CCCP, while those of 6.4 % (3/47) were decreased twofold.
.Mn53
DISCUSSION
Fig. 1. Dendrogram of PFGE profiles of the 32 erythromycin-nonsusceptible M. nonliquefaciens strains. Bar, similarity (%).
V00331) of M. nonliquefaciens ATCC 17953 (GenBank
accession no. AB818473), 18 different patterns of 23S
rRNA gene sequences were identified in the 47 M.
nonliquefaciens clinical isolates (GenBank accession nos
AB818474–AB818489). Although a large number of mutations were observed in the 32 erythromycin-non-susceptible and 15 erythromycin-susceptible strains, mutations
including A1986C, G1987A, A2058T, A2105G, T2113C,
T2132A, A2150T, T2151C, G2154A, C2160T and T2184C,
and an insertion at nt 2212 (E. coli numbering) were
detected only in the erythromycin-non-susceptible strains.
However, a single mutation, A2058T, was present only in
erythromycin-non-susceptible strains and was found in all
of these strains. The geometric mean MIC for erythromycin, clarithromycin, azithromycin and josamycin of 15
erythromycin-susceptible strains with the WT sequence at
http://jmm.sgmjournals.org
To date, although there are few case reports of M.
nonliquefaciens-associated infections, and M. nonliquefaciens has not yet been characterized in detail, including its
antimicrobial susceptibility. In general, Moraxella species
are susceptible to penicillin and its derivatives, cephalosporins, tetracyclines, quinolones, aminoglycosides, and
macrolides (Vaneechoutte et al., 2012). Furthermore,
previous studies have reported that M. nonliquefaciens is
susceptible to penicillin G, ampicillin, ceftazidime, tetracycline, ciprofloxacin, gentamicin, erythromycin and
imipenem (Davis et al., 2004; Laukeland et al., 2002). In
this study, most M. nonliquefaciens demonstrated low
penicillin MICs and showed extremely low MICs for
ampicillin, amoxicillin, ampicillin-sulbactam, amoxicillinclavulanic acid, cefixime and levofloxacin, as reported in
previous studies, while 68.1 % (32/47) of clinical isolates
demonstrated high-level MICs for 14-, 15- and 16-membered macrolides (e.g. erythromycin MIC .256 mg l21).
Therefore, although the present study has certain
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S. Nonaka and others
Table 3. MICs of macrolides in WT and A2058T mutant strains of M. nonliquefaciens
Antimicrobial
Erythromycin
Clarithromycin
Azithromycin
Josamycin
23S rRNA
sequence*
n
WT
A2058T
WT
A2058T
WT
A2058T
WT
A2058T
15
32
15
32
15
32
15
32
MIC (mg l”1)
Range
¡0.125–1
512–.512
¡0.125–0.5
128–.512
¡0.125
64–.512
0.5–4
128–256
MIC50
MIC90
¡0.125
512
¡0.125
512
¡0.125
256
2
256
0.5
512
0.5
512
¡0.125
512
4
256
P valueD
Geometric mean
0.229
513
0.201
378
0.125
235
2.09
185
,0.0001
,0.0001
,0.0001
,0.0001
*E. coli numbering (GenBank accession no. V00331).
DStatistical significance determined by a Mann–Whitney U test.
limitations in that sample numbers were small and medical
records of patients were not examined, our results indicate
that most M. nonliquefaciens clinical isolates in Japan
may have high-level resistance to macrolides, which are
frequently used for treatment of respiratory tract infections
such as pneumonia caused by Mycoplasma pneumoniae
(Grover et al., 2012; Higashi & Fukuhara, 2009; Kawai
et al., 2012). Furthermore, 32 erythromycin-non-susceptible
strains showed 30 banding pattern types by PFGE analysis,
indicating that various clones with high-level resistance to
macrolides are spreading throughout this country.
Recently, macrolide- and lincosamide-resistant M. catarrhalis strains have been reported in Japan (Saito et al.,
2012). Saito et al. (2012) demonstrated that the A2058T
mutation in the 23S rRNA gene (E. coli numbering) confers
high-level resistance to macrolides and lincosamides in M.
catarrhalis. Furthermore, it has been described that a single
mutation at position A2058 or A2059 in the 23S rRNA
gene confers high-level resistance to macrolides and/or
lincosamides in a variety of bacteria, including S.
pneumoniae, Mycobacterium smegmatis, Neisseria gonorrhoeae, Helicobacter pylori and M. pneumoniae (Chironna et
al., 2011; Chisholm et al., 2010; Li et al., 2011; TaitKamradt et al., 2000; Versalovic et al., 1997). In the present
study, although 32 erythromycin-non-susceptible strains
harboured various sequencing profiles in the 23S rRNA
gene, a single A2058T mutation was common to all
erythromycin-non-susceptible strains. Furthermore, the
geometric mean MIC for macrolides in A2058T-mutated
strains was significantly higher than that of WT strains
(P,0.0001). In contrast, erythromycin-non-susceptible
strains did not share common amino acid sequences in
L4 and L22, and erm genes were absent. Moreover, an
active efflux system was not only observed in three
erythromycin-non-susceptible strains, but also in one
erythromycin-susceptible strain. Therefore, although overexpression of efflux systems in addition to the A2058T
mutation in the 23S rRNA gene may contribute synergistically to the acquisition of resistance to macrolides, our
246
findings suggest the possibility that the presence of the
A2058T mutation is required to acquire high-level macrolide resistance in M. nonliquefaciens, as described previously for M. catarrhalis (Liu et al., 2012; Saito et al.,
2012). However, as we have not investigated the role of
the A2058T mutation in the 23S rRNA alleles of M.
nonliquefaciens to the same extent as in our previous M.
catarrhalis study (Saito et al., 2012), we propose to perform
further studies to analyse the role of the A2058T mutation
in more detail.
It is already known that M. nonliquefaciens produces BRO
b-lactamase (Eliasson et al., 1992). In this study, all 47 M.
nonliquefaciens clinical isolates carried bro b-lactamase
genes, and the frequency of bro-1- and bro-2-positive
isolates was 91.5 and 8.5 %, respectively. These observations are similar to previous findings of M. catarrhalis,
which is closely related to M. nonliquefaciens, in that more
than 90 % of the global clinical isolates are BRO blactamase producers, of which bro-1-positive isolates
make up a large majority (Khan et al., 2010; Murphy &
Parameswaran, 2009).
The present study demonstrates that M. nonliquefaciens
strains with high-level macrolide resistance have already
spread through clinical settings in Japan, but it is not clear
whether these results are related to the use of macrolides in
the treatment of infectious diseases, including respiratory
tract infections. Moreover, high-level macrolide-resistant
M. catarrhalis clinical isolates have already been reported in
Japan and China (Flamm et al., 2012; Liu et al., 2012; Saito
et al., 2012). To ensure rapid detection of the emergence
and prevention of further dissemination of high-level
macrolide-resistant phenotypes in Moraxella species, surveillance of antimicrobial susceptibility to macrolides
should be continued.
In conclusion, we demonstrate that 68.1 % of M. nonliquefaciens clinical isolates from Japan showed high-level
macrolide resistance and provide the first evidence that a
single A2058T mutation in the 23S rRNA gene confers this
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Journal of Medical Microbiology 63
Macrolide resistance in M. nonliquefaciens
high level of macrolide resistance. To our knowledge, this is
the first report investigating the role of mutations in the 23S
rRNA gene in M. nonliquefaciens. As the emergence and
spread of high-level macrolide-resistant Moraxella species
threatens effective treatment of these infections, continued
surveillance for these strains should be undertaken.
authors (2012). Clinical efficacy of macrolide antibiotics against
genetically determined macrolide-resistant Mycoplasma pneumoniae
pneumonia in paediatric patients. Respirology 17, 354–362.
Khan, M. A., Northwood, J. B., Levy, F., Verhaegh, S. J., Farrell, D. J.,
Van Belkum, A. & Hays, J. P. (2010). bro b-lactamase and antibiotic
resistances in a global cross-sectional study of Moraxella catarrhalis
from children and adults. J Antimicrob Chemother 65, 91–97.
Laukeland, H., Bergh, K. & Bevanger, L. (2002). Posttrabeculectomy
ACKNOWLEDGEMENTS
endophthalmitis caused by Moraxella nonliquefaciens. J Clin Microbiol
40, 2668–2670.
We would like to express our gratitude to Professor Norihisa
Noguchi who provided S. aureus, used as a positive control for
erm(A) and erm(C). The study did not receive financial support
from third parties. The authors declare that they have no conflicts of
interest.
Leclercq, R. & Courvalin, P. (1998). Streptogramins: an answer to
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