Download Emergence of a Streptococcus pneumoniae isolate resistant to

Journal of Antimicrobial Chemotherapy (2007) 59, 1010– 1012
doi:10.1093/jac/dkm041
Emergence of a Streptococcus pneumoniae isolate resistant to
streptogramins by mutation in ribosomal protein L22 during
pristinamycin therapy of pneumococcal pneumonia
Vincent Cattoir1*, Lilia Merabet1, Patrick Legrand1, Claude-James Soussy1 and Roland Leclercq2
1
Laboratoire de Bactériologie-Virologie-Hygiène, CHU Henri Mondor, Assistance Publique-Hôpitaux de Paris,
Université Paris XII, Créteil, France; 2Service de Microbiologie, EA 2128 Relations Hôte et Microorganismes
des Épithéliums, CHU Côte de Nacre, Université de Caen, 14033 Caen Cedex, France
Received 5 December 2006; returned 5 January 2007; revised 17 January 2007; accepted 29 January 2007
Objectives: The aim of this study was to characterize the mechanism of resistance to macrolides and
streptogramins of a Streptococcus pneumoniae strain isolated from blood cultures in an 80-year-old
patient suffering from severe pneumonia unsuccessfully treated with pristinamycin.
Methods: Resistance genes erm(B) and mef(A) were searched for by PCR. Portions of genes for
domains V and II of the 23S rRNA (rrl) and genes for ribosomal proteins L4 (rplD) and L22 (rplV) were
amplified by PCR from total genomic DNA and sequenced.
Results: Resistance genes erm(B) and mef(A) were not detected. Only mutation in the rplV gene encoding ribosomal protein L22 was detected. The strain contained a six amino acid insertion (107KRTAHI108)
in the C-terminus of the ribosomal protein L22.
Conclusions: This is the first report of emergence of a pneumococcus resistant to streptogramins by
mutation in ribosomal protein L22 during treatment with pristinamycin.
Keywords: macrolide resistance, pneumococcus, riboprotein, rplV
Introduction
Streptococcus pneumoniae is an important cause of communityacquired pneumonia. Penicillins are the treatment of choice of
these infections and macrolides constitute an alternative in the
case of intolerance to the drugs. However, increasing resistance
to macrolides and related antibiotics [macrolides– lincosamides –
streptogramins (MLS)] among S. pneumoniae clinical isolates
constitutes a worldwide problem.1 In pneumococci, MLS resistance is mediated by two major mechanisms: target site modification and drug efflux.1 Ribosomal alteration is mediated by a
methyltransferase, encoded by the erm(B) gene [or rarely the
erm(A) gene], that methylates the A2058 residue (Escherichia
coli numbering) in domain V of the 23S rRNA and confers the
so-called MLSB phenotype of cross-resistance among macrolides, lincosamides and streptogramins B. Active efflux is
mediated by the mef(A) gene [formerly mef(E)] that codes for
an efflux pump and is responsible for the so-called M phenotype
characterized by unique resistance to 14- and 15-membered-ring
macrolides (azithromycin, erythromycin, clarithromycin and
roxithromycin).1 In addition, mutations in 23S rRNA and the
ribosomal proteins L4 and L22 (encoded by the rplD and rplV
genes, respectively) have been infrequently described in pneumococci.2 Resistance to macrolides can reach percentages
exceeding 50% in certain countries and reduced susceptibility to
erythromycin has become more frequent than reduced susceptibility to penicillin in Europe overall.3 Telithromycin, a ketolide,
and pristinamycin, an oral streptogramin, are molecules related
to macrolides, which have demonstrated activity against pneumococci resistant to macrolides.4 Pristinamycin is a combination
of streptogramin A-type ( pristinamycin IIA) and streptogramin
B-type ( pristinamycin IA) antibiotics. Although Erm methylases
confer cross-resistance to macrolides, lincosamides and
streptogramins B, synergism between the two streptogramin
components is maintained, explaining the activity of the streptogramin combination.2 Here, we report an S. pneumoniae isolate
resistant to pristinamycin, an oral streptogramin, and mutated in
ribosomal protein L22 isolated from blood cultures of a patient
with lower respiratory tract infection unsuccessfully treated with
pristinamycin.
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*Corresponding author. Tel: þ33-1-49-81-68-16; Fax: þ33-1-49-81-28-39; E-mail: [email protected]
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S. pneumoniae L22 ribosomal mutant
Materials and methods
Table 1. MICs of MLS antibiotics for S. pneumoniae HM8989 and
for control strain, S. pneumoniae ATCC 49619
Bacterial strains and antimicrobial susceptibility testing
The strain of S. pneumoniae HM8989 was isolated from two blood
cultures taken from an 80-year-old male patient suffering from
severe pneumonia. He had been treated empirically with pristinamycin (3 g day) for 1 week for bronchial infection and was hospitalized
at Henri Mondor hospital (Créteil, France) for persistent fever and
increasing dyspnoea. The S. pneumoniae isolate was phenotypically
identified and antimicrobial susceptibility was tested by the disc diffusion method. MICs of MLS antibiotics were determined by the
agar dilution method on Mueller –Hinton agar medium, according
to the recommendations of Comité de l’Antibiogramme de la
Société Française de Microbiologie.5 The reference strain S. pneumoniae ATCC 49619 was included as a control.
MIC (mg/L)
Antibiotics
Erythromycin
Clarithromycin
Azithromycin
Spiramycin
Lincomycin
Clindamycin
Quinupristin
Dalfopristin
Quinupristin/
dalfopristin
Pristinamycin IA
Pristinamycin IIA
Pristinamycin
Telithromycin
PCR and DNA sequence analysis
Bacterial genomic DNA was extracted using the QIAmp DNA Mini
Kit (Qiagen, Courtaboeuf, France). Resistance genes erm(B) and
mef(A) and portions of genes for domains V and II of the 23S
rRNA (rrl) and genes for ribosomal proteins L4 (rplD) and L22
(rplV) were amplified using PCR with specific primers, as previously described.6 PCR products were then sequenced in both
directions using the ABI PRISM 3100 automatic sequencer
(Applied Biosystems, Courtaboeuf, France). For identification of
ribosomal mutations, sequences were compared with the genomic
sequences of S. pneumoniae TIGR4 and R6 (GenBank accession
numbers NC003098 and NC003028, respectively). Comparisons
were done using the software available over the Internet at the
National Center for Biotechnology Information web site (http://
www.ncbi.nlm.nih.gov/).
Results
Susceptibility to antimicrobial agents
By the disc diffusion method, the pneumococcal strain HM8989
was fully susceptible to all b-lactams tested with MICs of penicillin G, ampicillin and cefotaxime equal to 0.016, 0.016 and
0.008 mg/L, respectively. It also showed an intrinsic low-level
resistance to aminoglycosides (streptomycin, kanamycin and
gentamicin) and was susceptible to tetracyclines, chloramphenicol, rifampicin, co-trimoxazole, levofloxacin and vancomycin.
In contrast, it exhibited a very uncommon profile of resistance to
macrolides and related antibiotics with an intermediate susceptibility to erythromycin and pristinamycin. The strain remained
fully susceptible to lincomycin. No D-shaped zone was visible
when the disc of erythromycin was placed close to the disc of
clindamycin.
MICs of MLS antibiotics are shown in Table 1. MICs of
erythromycin, clarithromycin, azithromycin, telithromycin and
spiramycin for the strain HM8989 were 32– 64-fold greater than
those for the reference strain S. pneumoniae ATCC 49619.
Despite the 32-fold increased MIC of telithromycin, the isolate
remained categorized as susceptible to this antibiotic. MIC
determination confirmed that the isolate was fully susceptible to
lincosamides with MICs 2– 4-fold lower than those for the reference strain. In addition, the isolate was resistant to the streptogramin combination quinupristin/dalfopristin with an 8-fold
increase in the MIC of quinupristin, whereas the MIC of dalfopristin was the same. As expected, this strain was classified as
S. pneumoniae
HM8989
S. pneumoniae
ATCC 49619
1
1
2
8
0.12
0.03
8
32
16
0.03
0.016
0.06
0.12
0.5
0.06
1
32
0.25
16
4
2
0.25
4
2
0.12
0.008
intermediate to pristinamycin (MIC ¼ 2 mg/L), with MICs of
pristinamycin IA and pristinamycin IIA equal to 16 and 4 mg/L,
respectively.
Mutation in the rplV gene
No erm(B) methylase gene or mef(A) efflux gene could be
detected by PCR with specific primers. No mutations were found
in the rrl gene (domains II and V) or in the rplD gene for ribosomal protein L4. However, the strain displayed an 18 nucleotide
tandem duplication at the 30 end of the rplV gene encoding ribosomal protein L22. This insertion corresponded to a six amino
acid insertion (107KRTAHI108) tandemly duplicated (Figure 1).
Discussion
Macrolide resistance in S. pneumoniae is mostly mediated by
erm(B), a 23S rRNA methylase, and by mef(A), a gene encoding
an efflux pump.2 Since pristinamycin is still active against pneumococcal isolates displaying either mechanism,2 this oral antibiotic represents an attractive option for the empirical treatment
of respiratory tract infections. This report shows that resistance
to streptogramins may emerge in clinical isolates through
mutation in ribosomal protein L22 and can be associated with
clinical failure of pristinamycin therapy. No early pneumococcal
isolate from the patient was available. Therefore, we do not
know whether the patient was infected by a strain already intermediate resistant to pristinamycin or whether strain HM8989
was a mutant selected under pristinamycin therapy. A few cases
of clinical failure of pristinamycin for the treatment of lower respiratory tract infections have been reported.7,8 However, in these
episodes, the strains remained susceptible to pristinamycin. Only
two pneumococcal isolates from blood cultures have been
reported in 1989, resistant to 16-membered macrolides and intermediate to pristinamycin. This resistance was shown to be due
to an A2062C mutation in domain V of the 23S rRNA.9 Since
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Cattoir et al.
S. pneumoniae HM8989
10
20
30
40
50
60
70
....|....|....|....|....|....|....|....|....|....|....|....|....|....|
MAEITSAKAMARTVRVSPRKSRLVLDNIRGKSVADAIAILTFTPNKAAEIILKVLNSAVANAENNFGLDK
S. pneumoniae TIGR4
MAEITSAKAMARTVRVSPRKSRLVLDNIRGKSVADAIAILTFTPNKAAEIILKVLNSAVANAENNFGLDK
S. pneumoniae R6
MAEITSAKAMARTVRVSPRKSRLVLDNIRGKSVADAIAILTFTPNKAAEIILKVLNSAVANAENNFGLDK
S. pneumoniae HM8989
80
90
100
110
120
....|....|....|....|....|....|....|....|....|....|
ANLVVSEAFANEGPTMKRFRPRAKGSASPINKRTAHIKRTAHITVAVAEK
S. pneumoniae TIGR4
ANLVVSEAFANEGPTMKRFRPRAKGSASPIN------KRTAHITVAVAEK
S. pneumoniae R6
ANLVVSEAFANEGPTMKRFRPRAKGSASPIN------KRTAHITVAVAEK
Figure 1. Alignment of portion of the deduced amino acid sequence of ribosomal protein L22 of S. pneumoniae HM8989 when compared with those of
strains S. pneumoniae TIGR4 and R6.
then, resistance to pristinamycin in clinical isolates of pneumococci from France has not been reported.10 Mutations in protein
L22 seem to be uncommon in clinical isolates. In epidemiological surveys, mutations occurring in the 30 end of the
rplV gene have recently been shown to confer unusual
MLS-resistance patterns in clinical isolates [R22C, G95D, insertion 92VRPR93, A101P, tandem duplications (108RTAHI109 or
108
RTAHIT109), C117T].2 Emergence of an L22 mutant during
treatment with azithromycin of a fatal pneumococcal pneumonia
emphasizes the clinical importance of this mutation as a resistance mechanism.11 This isolate contained a tandem duplication
108
RTAHIT109 with resistance to erythromycin, azithromycin
and quinupristin/dalfopristin (MICs 2 – 4 mg/L). The structure of
the 50S ribosomal subunit containing a three-residue deletion
mutant of L22 in Haloarcula marismortui showed a change in
the L22 structure.11 L22 mutations at the C-terminus could
change the conformation of the b-hairpin and enlarge the exit
tunnel for polypeptides. This suggested that L22 mutants are
resistant to macrolides, because the lumen of the tunnel has
become so large that it can no longer be obstructed efficiently
by macrolides. However, as pointed out by Tu et al.,12 the mechanism of L22 resistance is likely to be more complex, since
macrolides bind to the tunnel entirely above the region affected
by L22 mutations. The mechanism of resistance by mutation in
L22 in S. pneumoniae has not been studied, but is probably
similar. The tandem duplication that we found in the C-terminus
of the L22 protein (107KRTAHI108) was not exactly the same as
those that were previously described (108RTAHIT109) among
clinical isolates of macrolide-resistant S. pneumoniae.2 However,
these two close variations could be responsible for similar conformational changes in the riboprotein. In the literature, there is
only one report of a macrolide-resistant pneumococcal mutant
emerging during empirical treatment. Finally, L22 mutations did
not affect the susceptibility to lincosamides but compromised
the potency of the ketolides, with an increase in MIC of telithromycin from 0.008 to 0.25 mg/L.
This report confirms the role of tandem duplications of ribosomal target protein L22 in MLS resistance in S. pneumoniae.
This is the first report of emergence of a pneumococcal isolate
resistant to streptogramins by mutation in ribosomal protein L22
during treatment with pristinamycin. Finally, it also highlights
the risk of emergence of these mutants that present increased
MICs of telithromycin.
Transparency declarations
None to declare.
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