In vitro activity of the new quinolone derivative RD-3 against clinical isolates of Mycoplasma pneumoniae and Mycoplasma hominis.

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genetic environment of qnrB2 has been described in the
Salmonella Enteritidis and Keurmassar serovars, as well as in
the Klebsiella strains and partially in C. koseri, being highly
similar in all bacteria described to date (Figure 1). In pB1004,
immediately upstream of qnrB2, an IS26 is followed by two
genes homologous to genes located in the chromosome of the
marine bacterium Marinobacter aquaeolei VT8 (GenBank
accession number CP000514). The intM gene encodes IntM, a
513 amino acid protein with 68.3% identity with Maqu_0026,
encodingthe catalyticdomain
Downstream of intM, istB codes for IstB, an ATP-binding
protein with 83.4% identity with Maqu_0025 of M. aquaeolei
involved in the transposition of IS21-like elements. The two
genes are located in the same order in the chromosome of
M. aquaeolei VT8. Interestingly, both genes are present in the
recently described Taiwanese InHI2 plasmids pEC-IMP and
pEC-IMPQ, the latter also bearing the qnrB2 gene. Further
analysis by PCR and sequencing of the genetic environment of
qnrB2 revealed the existence of an 8939 bp deletion including
an ISCR1 element and a class I integron with the blaIMP-8
metallo-b-lactamase gene.
Here we report the identification of a qnr gene in Salmonella
in Spain. Previously, qnrB2 has been identified in Enterobacter
spp. and qnrA has been detected in E. coli, E. cloacae and
K. pneumoniae, whereas the qnrS gene has been shown to be
present in a K. pneumoniae clinical isolate in this country. In
other countries like France, the UK, Germany, Israel, Australia,
the USA, Taiwan, the Netherlands and Senegal, the qnrB gene
is largely present.6In the latter four, Salmonella spp. was the
host bacterium of the qnrB2 gene. In Taiwan, the gene was
present in the serovar Enteritidis,4and possessed a similar
genetic environment to that described previously for Salmonella
Keurmassar.5Interestingly, the single qnrB2-bearing Salmonella
originating from a Dutch broiler chicken also belongs to serovar
Bredeney (isolate 137.25)6and was suspected to be potentially
linked to the Bredeney isolate from Spain. However, the genetic
environment flanking the qnrB2 gene and the plasmid incompat-
ibility groups are completely different for the two Bredeney iso-
lates from the Netherlands and Spain (IncN-p137.25 and
IncHI2-pB1004, respectively), suggesting independent events of
acquisition of qnrB2-carrying plasmids in these isolates.7
In contrast, the genetic environment of this qnrB2, as well as
the partial sequence of the plasmid backbone, reveals a striking
common evolutionary origin of pB1004 with pEC-IMPQ. This
is further supported by the plasmid size of a Taiwanese plasmid
that has been shown to be 324 kbp,2as compared with the
315 kbp from pB1004. It is tempting to speculate that the ISCR1
element has been responsible for the deletion of the 8939 bp in
pB1004 through rolling-circle replication. However, detailed
analysis of the junctions reveals that the deletion occurred
upstream of the replication origin of the ISCR1 element.
Further, a perfect IRL (‘left inverted repeat’) structure in the
IS26 element downstream of qnrB2 is evident in pB1004, indi-
cating that insertion of IS26 occurred after deletion of the
8939 bp fragment. These results confirm emergence in Spain of
pB1004, an IncHI2 plasmid related to pEC-IMPQ, that associ-
ates the qnrB2 gene with SHV-12 and TEM-1.
of theIS21transposase.
Acknowledgements
N. Montero is acknowledged for excellent technical assistance.
Funding
This work was supported by WP29 of the Network of
Excellence FOOD-CT-506122
MICINN (GEN2006-27767-E/PAT). J. A. E., A. S. M. and
L. H. acknowledge the Universidad Complutense de Madrid, the
Spanish Ministry of Education and Science and the Comunidad
de Madrid for their respective fellowships.
MED-VET-NETandthe
Transparency declarations
None to declare.
Supplementary data
Table S1 is available as Supplementary data at JAC Online
(http://jac.oxfordjournals.org/).
References
1. Hopkins KL, Day M, Threlfall EJ. Plasmid-mediated quinolone
resistance in Salmonella enterica, United Kingdom. Emerg Infect Dis
2008; 14: 340–2.
2. Chen YT, Liao TL, Liu YM et al. Mobilization of qnrB2 and ISCR1
in plasmids. Antimicrob Agents Chemother 2009; 53: 1235–7.
3. Jacoby GA, Walsh KE, Mills DM et al. qnrB, another plasmid-
mediated gene for quinolone resistance. Antimicrob Agents Chemother
2006; 50: 1178–82.
4. Wu JJ, Ko WC, Chiou CS et al. Emergence of Qnr determinants
in human Salmonella isolates in Taiwan. J Antimicrob Chemother
2008; 62: 1269–72.
5. Garnier F, Raked N, Gassama A et al. Genetic environment of
quinolone resistance gene qnrB2 in a complex sul1-type integron in the
newly described Salmonella enterica serovar Keurmassar. Antimicrob
Agents Chemother 2006; 50: 3200–2.
6. Veldman K, van Pelt W, Mevius D. First report of qnr genes
in Salmonella in The Netherlands. J Antimicrob Chemother 2008; 61:
452–3.
7. Fortini D, Garcı ´a-Ferna ´ndez A, Veldman K et al. Novel genetic
environment of plasmid-mediated quinolone resistance gene qnrB2 in
Salmonella Bredeney from poultry. J Antimicrob Chemother 2009; 64:
1332–4.
Journal of Antimicrobial Chemotherapy
doi:10.1093/jac/dkp375
Advance Access publication 21 October 2009
In vitro activity of the new quinolone derivative RD-3
against clinical isolates of Mycoplasma pneumoniae
and Mycoplasma hominis
Shilpakala Sainath Rao* and Malathi Raghunathan
Department of Genetics, Dr ALMPGIBMS, University
of Madras, Taramani Campus, Chennai 600113, India
Keywords: MICs, MBCs, fluoroquinolones
*Corresponding author. Tel: þ91-044-24925317;
Fax: þ91-044-24926709; E-mail: shilpa_ibms@yahoo.com
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Sir,
RD-3 is a new investigational quinolone derivative with
the formula bis(4,9,9a,10-tetrahydro-9-phenyl-3bH-pyrrolizino-
[1, 2-b]quinolin-7-)methane 12, synthesized by the Department
of Organic Chemistry, University of Madras, Chennai, India
(Figure 1). This drug has shown significant activity against
Gram-positive and Gram-negative organisms.1
Mycoplasma pneumoniae is a significant cause of upper and
lower respiratory tract infections in persons of all age groups.2
In some cases, these organisms can cause severe, systemic
disease, especially in the setting of a debilitated or immunocom-
promised host. Current treatment alternatives are limited primar-
ily to drugs in the macrolide, lincosamide, tetracycline and
fluoroquinolone classes, and agents in these classes exhibit
differential in vitro activities against these organisms. Though
macrolides are the treatment of choice for M. pneumoniae respir-
atory infections, the emergence of macrolide-resistant strains of
M. pneumoniae has been reported in Japan.3Mycoplasma
hominis most frequently causes diseases of the genitourinary
tract. In this study, we examined the activity of RD-3 against 61
clinical isolates of M. pneumoniae collected from patients with
pneumonia and 40 genital tract isolates of M. hominis. Ten of
the isolates were resistant to tetracycline (MIC90, ?8 mg/L).
The comparator agents moxifloxacin, ciprofloxacin, levoflox-
acin, gatifloxacin, gemifloxacin, azithromycin, doxycycline,
tetracyclineandclindamycin
Sigma-Aldrich (St Louis, MO, USA). Azithromycin was tested
only against M. pneumoniae since M. hominis is always resistant
to macrolides,2
and clindamycin was tested only against
M. hominis since it is not recommended for the treatment of
M. pneumoniae.4
Antimicrobial powders were used according to the manufac-
turer’s protocol. Working dilutions of the drugs were prepared
fresh on the day of the assay.
Mycoplasmas were tested by the agar dilution method using
a Steers replicator as described previously.2M. pneumoniae
ATCC 29342 and M. hominis ATCC 43521 were used as refer-
ence strains. The MICs of the compounds tested against the
reference strains for quality control were reproducible through-
out the study. The inoculum was derived from actively growing
cultures in broth medium supplemented with 20% serum and
10% fresh yeast extract.5The MIC was the least amount of anti-
microbial agent that completely prevented colony formation by
an inoculum of 30–300 cfu per spot, with a 5 day incubation for
M. hominis and a 14 day incubation for M. pneumoniae. The
growth control was subcultured to ensure the presence of viable
organisms in the absence of the drug. Broths were incubated at
378C. Ten isolates of M. pneumoniae were randomly chosen for
further testing to determine MBCs according to the protocol pre-
viously described.2The MBC was defined as the concentration
of the antimicrobial at which no growth was apparent, as shown
by lack of colour change in the broth after prolonged incubation.
MICs of RD-3 and other antimicrobial agents are shown in
Table 1. RD-3 and azithromycin were the drugs most active
against M. pneumoniae (MIC50, 0.001 mg/L; MIC90, 0.016 mg/L).
Gemifloxacin (MIC50, 0.03 mg/L; MIC90, 0.125 mg/L) was
more active than moxifloxacin (MIC50, 0.06 mg/L; MIC90,
0.125 mg/L), gatifloxacin (MIC50,
0.125 mg/L), doxycycline (MIC50, 0.25 mg/L; MIC90, 0.25 mg/L),
levofloxacin(MIC50, 1 mg/L;
were purchased from
0.125 mg/L;MIC90,
MIC90, 1 mg/L),
tetracycline (MIC50, 1 mg/L; MIC90, 1 mg/L) and ciprofloxacin
(MIC50,4 mg/L;MIC90,4 mg/L).
(MBC50, 0.001 mg/L; MBC90, 0.016 mg/L) and moxifloxacin
(MBC50, 0.06 mg/L; MBC90, 0.12 mg/L) showed that these
molecules were bactericidal against each of the 10 isolates of
M. pneumoniae tested.
M. hominis was highly susceptible to RD-3 (MIC50, 0.03 mg/L;
MIC90, 0.06 mg/L), (Table 1). RD-3 was as active as clindamycin
(MIC50, 0.03 mg/L), and was more active than moxifloxacin
(MIC50, 0.06 mg/L; MIC90, 0.06 mg/L), gatifloxacin (MIC50,
0.125 mg/L; MIC90, 0.125 mg/L), levofloxacin (MIC50, 0.25 mg/L;
MIC90, 0.5 mg/L), ciprofloxacin (MIC50, 1 mg/L; MIC90, 1 mg/L),
doxycycline (MIC50, 2 mg/L; MIC90, 2 mg/L) and tetracycline
(MIC50, 8 mg/L; MIC90, 8 mg/L).
Our study indicates that RD-3 is active in vitro against
Mycoplasma species that are clinically important in humans
with activity that was comparable to azithromycin and superior
to the fluoroquinolones tested. Earlier work demonstrated that
RD-3 inhibited gyrase supercoiling with potency similar to
ciprofloxacin and down-regulated gyrase A gene expression in
The MBCsof RD-3
N
N
H
N
PhH
Ph
H
H
N
Figure 1. Chemical structure of RD-3.
Table 1. Susceptibilities of M. pneumoniae and M. hominis to
RD-3 and other antimicrobial agents
Organism/drug
MIC (mg/L)
range50%a
90%b
M. pneumoniae (61)
RD-3
azithromycin
gemifloxacin
moxifloxacin
gatifloxacin
doxycycline
levofloxacin
tetracycline
ciprofloxacin
0.001–0.016
0.001–0.016
0.004–0.125
0.06–0.25
0.016–0.25
0.016–1
0.0125–2
0.25–1
0.5–8
0.001
0.001
0.03
0.06
0.125
0.25
1
1
4
0.016
0.016
0.125
0.125
0.125
0.25
1
1
4
M. hominis (40)
clindamycin
RD-3
moxifloxacin
gatifloxacin
levofloxacin
ciprofloxacin
doxycycline
tetracycline
0.008–0.5
0.016–0.25
0.06–0.25
0.016–0.25
0.125–1
0.25–2
0.008–2
0.5–16
0.03
0.03
0.06
0.125
0.25
1
2
8
0.03
0.06
0.06
0.125
0.5
1
2
8
aMICs at which 50% of the isolates tested were inhibited (MIC50).
bMICs at which 90% of the isolates tested were inhibited (MIC90).
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Escherichia coli.1These in vitro data indicate that RD-3 may
show considerable promise for both genital and respiratory
infections with mycoplasmas, but its clinical utility will depend
upon its toxicity and pharmacokinetics.6
Acknowledgements
We thank Professor Ragavachari Raghunathan, Department of
Organic Chemistry, University of Madras, for providing the
compound RD-3.
Funding
This study was funded in part by financial support from the
University of Madras, Chennai, India.
Transparency declarations
None to declare.
References
1. Ramesh E, Raghunathan R, Manian RD et al. Synthesis and anti-
bacterial property of quinolines with potent DNA gyrase activity. Bioorg
Med Chem 2009; 17: 660–6.
2. Waites KB, Taylor-Robinson D. Mycoplasma and Ureaplasma. In:
Murray PR, ed. Manual of Clinical Microbiology. Washington, DC: ASM
Press, 2007; 1004–20.
3. Morozumi MS, Iwata K, Hasegawa K et al. Increased macrolide
resistance of Mycoplasma pneumoniae in pediatric patients with
community-acquired pneumonia. Antimicrob Agents Chemother 2008;
52: 348–50.
4. Clyde WA Jr. Mycoplasma pneumoniae infections of man. In:
Tully JG, Whitcomb RF, eds. The Mycoplasmas II. Human and Animal
Mycoplasmas. New York: Academic Press, 1979; 275–306.
5. Kenny GE. Mycoplasmas. In: Balows A, Hausler WJ Jr,
Herrmann KL, eds. Manual of Clinical Microbiology. Washington, DC:
ASM Press, 1991; 478–82.
6. Domagala JM. Structure-activity and structure-side-effect relation-
ships for the quinolone antibacterials. J Antimicrob Chemother 1994;
33: 685–706.
Journal of Antimicrobial Chemotherapy
doi:10.1093/jac/dkp376
Advance Access publication 14 October 2009
Susceptibility of pneumococci causing meningitis
in Spain and prevalence among such isolates of
serotypes contained in the 7-valent pneumococcal
conjugate vaccine
Asuncio ´n Fenoll1, Marı ´a-Jose ´ Gime ´nez2,
Marı ´a-Dolores Vicioso1, Juan-Jose ´ Granizo3,
Olga Robledo1and Lorenzo Aguilar2*
1Spanish Reference Pneumococcal Laboratory, Instituto
Salud Carlos III, Majadahonda, Madrid, Spain;
2Microbiology Department, School of Medicine, Universidad
Complutense, Madrid, Spain;3Grana Datos S.L., Pozuelo de
Alarco ´n, Madrid, Spain
Keywords: laboratory-based surveillance, cerebrospinal fluid,
Streptococcus pneumoniae
*Corresponding author. Tel: þ34-91-3941505;
Fax: þ34-91-3941511; E-mail: laguilar@med.ucm.es
Sir,
Streptococcus pneumoniae remains the most common cause of
bacterial meningitis in children in the USA,1although since the
introduction of the 7-valent pneumococcal conjugate vaccine
(PCV-7) rates have decreased despite the increase in meningitis
caused by non-PCV-7 serotypes.2In Spain the decrease in the
prevalence of PCV-7 serotypes among the global population of
invasive pneumococci after the introduction of PCV-7 was not as
marked as in the USA, probably due to irregular and lower cover-
age. After vaccine introduction in 2001, distribution was via the
private market because of the selective introduction into child-
hood vaccination calendars (it was only introduced in the Madrid
region from November 2006).3,4A recently published ecological
analysis of invasive isolates over time in Spain suggested that
PCV-7 vaccination in children had produced a herd effect (with
respect to prevalence of PCV-7 isolates and antibiotic suscepti-
bility) in adults.3It has also been reported that the incidence of
pneumococcal meningitis among children ,5 years old signifi-
cantly decreased in Spain from 2001 to 2006, without evidence
of changes in the incidence of meningitis caused by non-vaccine
serotypes.5Although a small proportion of invasive pneumococ-
cal infections present as meningitis, it has a high case-fatality
rate. Whether the empirical use of cefotaxime for meningitis
needs to be continued in countries with increasing PCV-7 uptake
needs to be monitored. We considered isolates from CSF
received in the Spanish Reference Laboratory for Pneumococci
(SRLP) in the current decade to analyse their susceptibility and
prevalence of PCV-7 serotypes.
All CSF isolates of S. pneumoniae sent voluntarily from all
over the country to the SRLP (passive, laboratory-based surveil-
lance system) from January 2000 to December 2008 were ana-
lysed. Isolates were serotyped by Quellung reaction and/or dot
blot assay, and susceptibility was determined by agar dilution.3,4
Current CLSI meningitis susceptibility breakpoints for penicillin
(MIC?0.06 mg/L) and cefotaxime (MIC?0.5 mg/L), and sus-
ceptibility breakpoints of MIC?1 mg/L for vancomycin and
MIC?2 mg/L for levofloxacin were used.6Trends over time
were explored by linear regression analysis. P?0.05 was con-
sidered significant.
Data are shown in Table 1. Of the 1397 CSF isolates received
between January 2000 and December 2008, 923 (66.1%) were
from adults and 474 (33.9%) from children ?14 years of age.
No significant trends in the percentage of CSF isolates among
invasive isolates were found in the three populations: total popu-
lation (R2¼0.008, P¼0.823), adults (R2¼0.240, P¼0.180) and
children (R2¼0.395, P¼0.070), although in children there was
a continuous decrease from 15.5% in 2003 to 9.8% in 2008.
The prevalence of PCV-7 serotypes among CSF isolates
showed significant decreasing linear trends in the total popu-
lation (R2¼0.914, b¼20.956, P,0.001), adults (R2¼0.819,
b¼20.905, P,0.001) and children (R2¼0.870, b¼20.933,
P,0.001), with a significantly higher decreasing slope in chil-
dren than in adults (B coefficient¼26.307, 95% CI¼28.485 to
24.128in childrenversus
CI¼24.963 to 22.027 in adults).
B coefficient¼23.495,95%
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