Letters to the Editor / International Journal of Antimicrobial Agents 43 (2014) 474–483 479

Table 1Correlation between susceptibility to fluoroquinolones and mutations in GrlA, GrlB, GyrA and GyrB in meticillin-resistant Staphylococcus aureus (MRSA).

Group Mutations No. of strains MIC range (�g/mL)

GrlA GrlB GyrA GyrB LVFX PZFX MFLX STFX

Susceptible –a – – – 2 0.25 0.25 ≤0.06 ≤0.06A S80F – S84L or E88G – 8 4–16 4–16 1–4 0.25–1B S80Y + E84K – S84L – 1 64 16 8 2C S80Y + E84K – S84L + E88G – 14 512 256–512 64 8–16D S80Y + E84K – S84L + E88Rb – 2 512 512 128 16–32E S80Y + E84K – S84L + E88G E477Db 8 512 512 128 32

M FLX, m

dGa

hsm1N(1siitistt

mbhtqpifsq

s

R

[

[

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IC, minimum inhibitory concentration; LVFX, levofloxacin; PZFX, pazufloxacin; Ma No mutation found.b Novel mutation discovered in this study.

ata strongly suggest that mutations E88R in GyrA and E477D inyrB confer high-level third-generation quinolone resistance to S.ureus.

To verify the influence of the novel E477D mutation in GyrB,igh-level third-generation quinolone-resistant strains were con-tructed in vitro using three mutant group C strains. High-leveloxifloxacin-resistant mutants were obtained at frequencies of

.0 × 10−10 to 2.1 × 10−9. Five mutants [E477D in GyrB (mutant 1),475D in GyrB (mutant 2), D83N in GyrA (mutant 3), S85P in GyrA

mutant 4) and G271N in GyrA (mutant 5)] were found. Mutanthad identical mutations to the clinical moxifloxacin-resistant

trains and the MICs of nadifloxacin, moxifloxacin and sitafloxacinn mutant 1 were two to eightfold higher than those in the orig-nal strain. Although mutants 2, 3, 4 and 5 had novel mutationshat were not observed in the clinical isolates, they exhibited sim-lar fluoroquinolone susceptibility to mutant 1. Therefore, the datatrongly support that the novel additional mutations in GyrB andhe QRDR region of GyrA are involved in the high-level resistanceo third-generation quinolones that have a dual-targeting action.

In conclusion, the mutation E477D in GyrB is the commonestutation for high-level third-generation quinolone resistance

ecause the mutation was found in eight clinical isolates exhibitingigh-level resistance to moxifloxacin. Previously, we demonstratedhat mutations in GyrB affect the resistance level to fluoro-uinolones in Helicobacter pylori [5]. Together, the current andrevious data strongly suggest that the mutation of GyrB is involved

n high-level resistance to third-generation quinolones. Furtherunctional analysis of the E477D mutation in GyrB is neces-ary to understand how it confers resistance to third-generationuinolones.

Funding: This work was supported by the Matching Fund Sub-idy for Private Schools of Japan.

Competing interests: None declared.Ethical approval: Not required.

eferences

1] Adriaenssens N, Coenen S, Versporten A, Muller A, Minalu G, Faes C, et al. ESACProject Group, European Surveillance of Antimicrobial Consumption (ESAC):outpatient quinolone use in Europe (1997–2009). J Antimicrob Chemother2011;66(Suppl. 6):vi47–56.

2] Takei M, Fukuda H, Kishii R, Hosaka M. Target preference of 15quinolones against Staphylococcus aureus, based on antibacterial activ-ities and target inhibition. Antimicrob Agents Chemother 2001;45:3544–7.

3] Okumura R, Hirata T, Onodera Y, Hoshino K, Otani T, Yamamoto T. Dual-targetingproperties of the 3-aminopyrrolidyl quinolones, DC-159a and sitafloxacin,against DNA gyrase and topoisomerase IV: contribution to reducing in vitroemergence of quinolone-resistant Streptococcus pneumoniae. J AntimicrobChemother 2008;62:98–104.

4] Hooper DC. Fluoroquinolone resistance among Gram-positive cocci. Lancet

Infect Dis 2002;2:530–8.

5] Rimbara E, Noguchi N, Kawai T, Sasatsu M. Fluoroquinolone resistance in Heli-cobacter pylori: role of mutations at position 87 and 91 of GyrA on the levelof resistance and identification of a resistance conferring mutation in GyrB.Helicobacter 2012;17:36–42.

oxifloxacin; STFX, sitafloxacin.

Hidemasa NakaminamiKoko Sato-Nakaminami

Norihisa Noguchi ∗

Department of Microbiology, School of Pharmacy, Tokyo Universityof Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo

192-0392, Japan

∗ Corresponding author. Tel.: +81 426 76 5619;fax: +81 426 76 5647.

E-mail address: noguchin@toyaku.ac.jp (N. Noguchi)

2 October 2013

http://dx.doi.org/10.1016/j.ijantimicag.2014.02.002

Revised and updated multiplex PCR targetingacquired 16S rRNA methyltransferases

Sir,

The increasing burden of resistance to most available antimi-crobial agents, especially in nosocomial Gram-negative infections,has renewed clinical interest in previously abandoned aminoglyco-sides. Resistance to these compounds may result from enzymaticinactivation, changes in drug uptake and efflux, and modificationof the target binding site. The latter mechanism is mediated by16S rRNA methyltransferases in clinically relevant Gram-negativerods and confers class-wide resistance to aminoglycosides [1].To date, 10 acquired 16S rRNA methyltransferase-encoding geneshave been identified, including armA, rmtA, rmtB, rmtC, rmtD, npmA,rmtE, rmtF, rmtG and rmtH [2].

Early detection of resistance genes may assist the implemen-tation of effective antimicrobial therapy. Multiplex methodologiestargeting 16S rRNA methyltransferases have been reported previ-ously [3,4], enabling the detection of armA, rmtA, rmtB, rmtC, rmtDand npmA. However, an attempt to amplify rmtD2 from positivecontrols in our collection yielded negative results (data not shown).This result might be explained by the presence of mismatches foundbetween rmtD2 and both reverse and forward primers. This finding,allied to the report of four additional 16S rRNA methyltransferase-encoding genes that are not amplified using the available platform,motivated the present work. Here we present an updated multiplexPCR scheme able to detect all of the acquired methyltransferasegenes described so far.

The scheme included two multiplex reactions with primersequences targeting armA, rmtA, rmtB, rmtC and npmA genes, previ-ously published by Bercot et al. [4], together with oligonucleotides

designed in the present study based on sequences availableat GenBank (accession nos. DQ914960, HQ401565, GU201947,JQ808129, JX486113 and KC544262) hybridising at rmtD/rmtD2,rmtE, rmtF, rmtG and rmtH genes, respectively. Nucleotide

480 Letters to the Editor / International Journal of Antimicrobial Agents 43 (2014) 474–483

Fig. 1. Electrophoresis profiles of multiplex PCR products. (A) Lanes 1 and 8, 100 bp Plus DNA Ladder; lane 2, rmtA; lane 3, rmtC; lane 4, rmtG; lane 5, rmtH; lane 6, rmtD;and lane 7, negative control. (B) Lanes 1 and 8, 100 bp Plus DNA Ladder; lane 2, armA; lane 3, rmtB; lane 4, npmA; lane 5, rmtF; lane 6, rmtE; and lane 7, negative control. PCRproduct sizes are indicated in base pairs.

al of A

sMsttwstN(AptbmasaBA8TCrGGM3TrC353

iri

oD(accce

p(m(g

ftiOefiltsoh

[

[

[

[

[

as multidrug-resistant (MDR) TB [1]. In addition to Mycobacteriumtuberculosis, there are other mycobacterial species that are oppor-

Letters to the Editor / International Journ

equences of rmtD and rmtD2 were aligned using Lasergene®

egAlignTM v.7.0 (DNASTAR, Inc., Madison, WI), and the consen-us regions were considered for primer design. The Primer-BLASTool available at the National Center for Biotechnology Informa-ion (NCBI) (http://www.ncbi.nlm.nih.gov/tools/primer-blast/)as used to obtain eligible oligonucleotide sequences. Pos-

ible hairpins and primer–dimers were evaluated withhe AutoDimer Tools and Hairpin [5] available at theational Institute of Standards and Technology website

http://yellow.nist.gov:8444/dnaAnalysis/primerToolsPage.do).utoDimer analysis was conducted considering the followingarameters: 1 M monovalent salt; 1 �M total strand concentra-ion; 57 ◦C temperature; and total score 3, which is determinedy combining the number of Watson–Crick base pairs (+1) withismatches (−1). Oligonucleotides were evaluated separately

nd then in their corresponding multiplex. Only those primershowing similar melting temperature (Tm) and no significantnnealing in 3′-OH ends were synthesised (Invitrogen, São Paulo,razil). Multiplex 1 consisted of primers targeting rmtA (F, 5′-AACTATTCCGCATGGTTC-3′/R, 5′-TCATGTACACAAGCTCTTTCC-3′;8 bp), rmtC (F, 5′-CAGGGGTTCCAACAAGT-3′/R, 5′-AGAGTATA-AGCTTGAACATAAGTAGA-3′; 246 bp), rmtD (F, 5′-TCGTTTCAG-ACGTAAAACA-3′/R, 5′-CAGCGCGAAATTCAAAAAGG-3′; 652 bp),mtG (F, 5′-ACGGAATGCCGCGCGAAGTA-3′/R, 5′-TCTCCGCAAGCA-ATCGCCG-3′; 381 bp) and rmtH (F, 5′-ATGACCATTGAACA-GCAGC-3′/R, 5′-AGGGCAAAGGTAAAATCCCA-3′; 464 bp).ultiplex 2 targeted armA (F, 5′-ATTTTAGATTTTGGTTGTGGC-

′/R, 5′-ATCTCAGCTCTATCAATATCG-3′; 101 bp), npmA (F, 5′-GGGC-ATCTAATGTGGTG-3′/R, 5′-TTTTTATTTCCGCTTCTTCGT-3′; 229 bp),mtB (F, 5′-ACTTTTACAATCCCTCAATAC-3′/R, 5′-AAGTATATAAGTT-TGTTCCG-3′; 171 bp), rmtE (F, 5′-GATGCCGTGTCTGTTACGCCG-′/R, 5′-ACGTGAACCCACGAGTCCTGC-3′; 446 bp) and rmtF (F,′-CGATCCTACTGGGCTCCAT-3′/R, 5′-GGCATAGTGCTTTTCCATGC-′; 314 bp).

Positive controls for PCRs included seven Escherichia coli TOP10solates harbouring plasmid vectors carrying armA, rmtA, rmtB,mtC, rmtE, rmtF and npmA as well as three Klebsiella pneumoniaesolates carrying rmtD2, rmtG and rmtH.

DNA was obtained by suspending each control strain in 500 �Lf pure sterile water up to 0.5 McFarland turbidity. Then, 1 �L of theNA suspension was added to 12.5 �L of GoTaq® Green Master Mix

Promega, São Paulo, Brazil), 2 pM of each corresponding primernd 1.5 �L of water, resulting in a total 25 �L reaction mixture. Theycling parameters were the same for both multiplex reactions andonsisted of initial denaturation at 94 ◦C for 10 min, followed by 25ycles of 94 ◦C for 45 s, 57 ◦C for 30 s and 72 ◦C for 25 s, and a finalxtension step at 72 ◦C for 3 min.

Each positive control was successfully amplified by its specificrimer and showed no cross-reaction with other oligonucleotidesFig. 1). However, the multiplex approach also managed to detect

ethyltransferase-positive strains from our laboratory collectionsdata not shown), proving to be a useful tool in tracing theseenes.

Since its first description in 2003, reports of methyltrans-erases have been made available worldwide. Of great concern isheir association with other resistance genes, mainly those encod-ng extended-spectrum �-lactamases, metallo-�-lactamases andXA-type �-lactamases, in multidrug-resistant pathogens. Theselements are frequently present in transposons within trans-erable plasmids belonging to distinct incompatibility groups,ncluding those of broad-host range [1,3]. This scenario high-ights the contribution of horizontal gene transfer in acceleratinghe dissemination of 16S rRNA methyltransferase genes andupports the need for tools allowing the rapid recognition

f these elements, such as the multiplex scheme proposedere.

ntimicrobial Agents 43 (2014) 474–483 481

Acknowledgments

The authors thank Dr. Yoshichika Arakawa and Jun-ichi Wachino(armA, rmtB, rmtC and npmA), Dr. Margaret A. Davis (rmtE), Dr.Patrice Courvalin (rmtF) and Dr. Patrick McGann (rmtH) for kindlyproviding positive control plasmids and strains.

Funding: The authors wish to thank Fundacão de Amparo àPesquisa do Estado do Rio de Janeiro (FAPERJ) for financial support[process no. E-26/11.780/2012] and the National Council forScientific and Technological Development (CNPq) for providing ascholarship to LLC.

Competing interests: None declared.

Ethical approval: Not required.

References

1] Wachino J, Arakawa Y. Exogenously acquired 16S rRNA methyltransferasesfound in aminoglycoside-resistant pathogenic Gram-negative bacteria: anupdate. Drug Resist Updat 2012;15:133–48.

2] O’Hara JA, McGann P, Snesrud EC, Clifford RJ, Waterman PE, Lesho EP, et al.Novel 16S rRNA methyltransferase RmtH produced by Klebsiella pneumoniaeassociated with war-related trauma. Antimicrob Agents Chemother 2013;57:2413–16.

3] Doi Y, Arakawa Y. 16S ribosomal RNA methylation: emerging resistance mech-anism against aminoglycosides. Clin Infect Dis 2007;45:88–94.

4] Bercot B, Poirel L, Nordmann P. Updated multiplex polymerase chain reaction fordetection of 16S rRNA methylases: high prevalence among NDM-1 producers.Diagn Microbiol Infect Dis 2011;71:442–5.

5] Vallone PM, Butler JM. AutoDimer: a screening tool for primer–dimer and hairpinstructures. Biotechniques 2004;37:226–31.

Laís Lisboa CorrêaLara Feital Montezzi

Raquel Regina BonelliBeatriz Meurer Moreira

Renata Cristina Picão ∗

Laboratório de Investigacão em Microbiologia Laboratório Integradode Microbiologia (LIM), Instituto de Microbiologia, Universidade

Federal do Rio de Janeiro, Rio de Janeiro, Brazil

∗ Corresponding author. Present address: Centro de Ciências daSaúde, Bloco I, Av. Carlos Chagas Filho 373, Instituto de

Microbiologia, Universidade Federal do Rio de Janeiro, CidadeUniversitária, Rio de Janeiro, RJ 21941-902, Brazil. Tel.: +55 21

2560 8344; fax: +55 21 2560 8344.E-mail addresses: renata.picao@micro.ufrj.br,

renata.picao@gmail.com (R.C. Picão)

20 February 2014

http://dx.doi.org/10.1016/j.ijantimicag.2014.02.003

Activity of 4,5-dihydro-1H-pyrazoles againstMycobacterium tuberculosis and nontuberculousmycobacteria

Sir,

Tuberculosis (TB) is considered a neglected disease. The littleinvestment in research towards the development of new drugshas recently changed as a result of the gravity and danger thatTB represents, with high rates of morbidity and mortality as well

tunistic pathogens, and the number of cases of infections due tothese nontuberculous mycobacteria (NTM) is increasing [2]. In this