Optochin Resistance in Streptococcus Pneumoniae: Mechanism, Signiﬁcance, and Clinical Implications

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Optochin Resistance in Streptococcus pneumoniae: Mechanism, Signiﬁcance, and Clinical Implications

Andreas Pikis,1,2 Joseph M. Campos,3,4,5,6 1Vaccine and Therapeutic Development Section, Oral Infection William J. Rodriguez,2,4,a and Jerry M. Keith1 and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland; Departments of 2Infectious Diseases and 3Laboratory Medicine, Children’s National Medical Center, and Departments of 4Pediatrics, 5Pathology, and 6Microbiology/Tropical Medicine, George Washington

University Medical Center, Washington, DC Downloaded from https://academic.oup.com/jid/article/184/5/582/808236 by guest on 23 September 2021

Traditionally, Streptococcus pneumoniae is identified in the laboratory by demonstrating susceptibility to optochin. Between 1992 and 1998, 4 pneumococcal isolates exhibiting optochin resistance were recovered from patients at Children’s National Medical Center. Three of the 4 isolates consisted of mixed populations of optochin-resistant and -susceptible organ- isms. Both subpopulations had identical antibiograms, serotypes, and restriction fragment profiles. The other isolate was uniformly resistant to optochin. Resistant strains had MICs of optochin 4–30-fold higher than susceptible strains, belonged to different serotypes, and had dissimilar restriction fragment profiles, indicating clonal unrelatedness. Resistance arose from single point mutations in either the a-subunit (W206S) or the c-subunit (G20S, M23I, and A49T) of H+-ATPase. There is speculation of a possible association between exposure to antimalarial drugs and evolution of optochin resistance. a-Hemolytic streptococci resistant to optochin, particularly invasive isolates, should be tested for bile solubility or with an S. pneumoniae DNA probe before identification as viridans streptococci.

Streptococcus pneumoniae remains a major cause of human twentieth century as a chemotherapeutic agent for the treatment morbidity and mortality, particularly at both extremes of the of lobar pneumonia. However, serious side effects coupled with age spectrum [1]. The recent emergence of pneumococci resis- treatment failures quickly terminated the therapeutic use of this tant to penicillin, third-generation cephalosporins, and other agent [5]. Although optochin susceptibility was first described antimicrobial agents has raised concerns about treatment of for differentiating pneumococci from other a-hemolytic strep- patients with serious pneumococcal infections [2]. Because of tococci in 1915 [6], the test was virtually unused by laboratories the increasing frequency of antimicrobial resistance, accurate until the mid-1950s [7–9]. During the first 30 years of its use, identification and antimicrobial susceptibility testing are crucial there were no reports of optochin-resistant pneumococci. Since for correct diagnosis and treatment of patients. then, there have been sporadic reports of optochin-resistant Differentiation of S. pneumoniae from other viridans strep- pneumococci [10–12]. Investigators from Spain have cloned, tococci depends on demonstrating optochin susceptibility, bile sequenced, and characterized Hϩ-ATPase c-subunit as the gene solubility, reaction with a specific DNA probe, or detection of encoding the optochin determinant [13]. species-specific capsular polysaccharides [3]. Most clinical mi- In the United States, only 2 optochin-resistant strains have crobiology laboratories today depend on the optochin suscep- been reported. We reported the first isolate in 1997 from Wash- tibility test [4]. ington, DC [14], and Borek et al. [15] reported another from Optochin, a quinine analogue, was introduced early in the Chicago later that year. We since have recovered 3 additional optochin-resistant strains. The purpose of this communication Received 12 March 2001; revised 21 May 2001; electronically published is to report our characterization of the optochin resistance phe- 26 July 2001. notype of these strains and to alert physicians and clinical mi- Presented in part: American Pediatric Society and Society for Pediatric crobiologists to the existence of these strains in the community. Research meeting, Washington, DC, May 1997 (abstract 755; Pediatr Res 1997; 41:128A); Pediatric Academic Societies and American Academy of Pediatrics joint meeting, Boston, May 2000 (abstract 1613; Pediatr Res 2000; 47:273A). Materials and Methods a Present affiliation: Center for Drug Evaluation and Research, Food and Drug Administration, Rockville, Maryland. Patients and strains. From 1 June 1992 through 31 May 1998, Reprints or correspondence: Dr. Andreas Pikis, National Institute of Den- 587 pneumococcal isolates were recovered from ordinarily sterile tal and Craniofacial Research, National Institutes of Health, Bldg. 30, Rm. 528, 30 Convent Dr., MSC 4350, Bethesda, MD 20892-4350 (apikis@dir body sites of patients treated at Children’s National Medical Center .nidcr.nih.gov). (Washington, DC). Three (0.5%) isolates were either uniformly resistant to optochin (strain 310) or consisted of mixed populations The Journal of Infectious Diseases 2001;184:582–90 ᭧ 2001 by the Infectious Diseases Society of America. All rights reserved. of optochin-susceptible and -resistant colonies (strains 606a and 0022-1899/2001/18405-0009$02.00 654; figure 1). An additional isolate exhibiting optochin-resistant JID 2001;184 (1 September) Optochin Resistance in S. pneumoniae 583 Downloaded from https://academic.oup.com/jid/article/184/5/582/808236 by guest on 23 September 2021

Figure 1. Optochin test (disk diffusion method) of Streptococcus pneumoniae isolates 310 and 654. Isolate 310 had no inhibitory zone around the optochin disk. Isolate 654 had a 114-mm inhibitory zone with optochin-resistant colonies within the zone. Isolates 606a and HV109 had results similar to those for isolate 654. variants (strain HV109) was recovered from the nasopharynx of a from overnight 5% sheep blood agar cultures. The suspensions were patient during a study that investigated the prevalence of penicillin- divided into 2 tubes. Deoxycholate reagent (2%; 0.5 mL; Becton resistant S. pneumoniae colonization in human immunodeficiency Dickinson Microbiology Systems) was added to one tube, and nor- virus–infected children. Isolates with optochin-resistant variants mal saline (0.5 mL) was added to the other. Tubes were incubated formed distinct colonies within the 14-mm diameter inhibitory zone at room temperature for 15 min. Complete clearing of the suspension that differentiates optochin susceptibility from resistance. Subcul- in the deoxycholate tube was indicative of S. pneumoniae. tures of these colonies exhibited uniform resistance to optochin. Serotyping. Serotyping was done with the Quellung (Neufeld) We used 2 optochin-susceptible clinical isolates and an unencap- reaction (capsular swelling) test. sulated reference strain (R6) as controls. Table 1 shows the char- Genomic DNA analysis. Preparation of agarose plugs containing acteristics of the study isolates. genomic DNA and analysis by pulsed-field gel electrophoresis Antibiotic susceptibility testing. MICs of optochin and several (PFGE) after digestion with SmaI were done as described elsewhere antimicrobial agents were determined by the agar dilution method. [16]. Bacterial suspensions were prepared in 0.9% NaCl from overnight Polymerase chain reaction (PCR). Based on the sequence re- cultures on 5% sheep blood agar and were adjusted to match the ported by Fenoll et al. [13], we designed primers 663 (5-TCGA- 0.5 McFarland turbidity standard. Suspensions were inoculated AAAGTGGATCAACAACTATCC-3) and 1016 (5-TGGGAAA- onto Mueller-Hinton agar containing 3% lysed horse blood and GAAGAAGTAACAAACTCG-3) to amplify the DNA fragment varying concentrations of optochin or selected antimicrobial encoding the ATPase c-subunit from the pneumococcal strains ex- Њ agents. Cultures were incubated at 35 C for 20–24 h in a 5% CO2 amined in this study. The components of the amplification mixtures atmosphere. The MIC was defined as the lowest concentration of (100 mL) were as follows: 5 U of Pfu DNA polymerase (Stratagene), optochin or antimicrobial agent that inhibited visible growth of 1ϫ reaction buffer provided by the manufacturer, 20 mM each of the isolate. the 4 dNTPs, 100 ng of DNA, and 250 ng of each primer. Ampli- Optochin susceptibility test by disk diffusion method. Optochin fication was done in a thermal cycler (model 9600; Perkin-Elmer disks (6 mm; Becton Dickinson Microbiology Systems) were ap- Instruments). After an initial 2-min incubation at 95ЊC, the mixture plied to trypticase soy agar plates with 5% sheep blood streaked was subjected to 30 cycles of amplification under the following con- with the organism being tested. After overnight incubation at 35ЊC ditions: 95ЊC, 1 min; 58ЊC, 1 min; 72ЊC, 2 min; and a final 10-min Њ ina5%CO2 atmosphere, inhibitory zones around the disk were runoff at 72 C. After purification, the PCR product was ligated into measured. Isolates displaying zones у14 mm in diameter were iden- pCR-Blunt vector (Invitrogen), and the ligation products were trans- tified as S. pneumoniae. Isolates displaying zones !14 mm were formed into Escherichia coli TOP10 competent cells. E. coli trans- tested for bile solubility. formants were selected on LB agar containing 50 mg/mL kanamycin. Bile solubility. Heavy bacterial suspensions (1 mL) matching the DNA sequence analysis. Sequencing was done with the di- 2.0 McFarland turbidity standard were prepared in normal saline deoxynucleotide chain-termination method by using modified T7 584 Pikis et al. JID 2001;184 (1 September)

Table 1. Clinical and laboratory characteristics of pneumococcal strains tested.

MIC, mg/mL Optochin Strain susceptibility Serotype Isolation site Optochin Penicillin Cefotaxime Chloramphenicol Vancomycin R6 S NA NA 2 р0.015 р0.015 1 0.25 13 S 23F Synovial ﬂuid 2 0.5 0.5 16 0.25 92 S 6A Blood 2 р0.015 р0.015 4 0.25 310 R 9V Blood 8 р0.015 р0.015 4 0.5 606aa S 14 CSF 2 р0.015 р0.015 4 0.5 R 14 CSF 64 р0.015 р0.015 4 0.5 654a S 19F Blood 2 0.5 0.5 16 0.25 R 19F Blood 16 0.5 0.5 16 0.25 Downloaded from https://academic.oup.com/jid/article/184/5/582/808236 by guest on 23 September 2021 HV109a S 6B Nasopharynx 2 р0.015 р0.015 2 0.25 R 6B Nasopharynx 32 р0.015 р0.015 2 0.25 NOTE. CSF, cerebrospinal ﬂuid; NA, not applicable; R, resistant; S, susceptible. a Strains with optochin-resistant variants.

DNA polymerase and [a-35S]deoxyadenosine triphosphate for la- sistant colonies. All optochin-susceptible strains and, surpris- beling. For all clones, both strands of inserts were sequenced. We ingly, 1 resistant strain (HV109/R) possessed identical ATPase used the MacVector 7.0 sequence analysis package (Genetics Com- c-subunit amino acid sequences. The amino acid sequences in puter Group) to assemble, edit, and analyze the results. the 3 remaining optochin-resistant strains differed from the sus- Genetic transformation of S. pneumoniae. S. pneumoniae was ceptible strains by only 1 amino acid. Specifically, isoleucine genetically transformed according to a procedure described by replaced methionine at residue 23 in resistant strain 310, threo- Lacks [17], with the following modification: growth medium was supplemented with 0.2% sucrose, but yeast extract was omitted. nine replaced alanine at residue 49 in resistant strain 606a/R, Transformants were selected on Mueller-Hinton agar plates con- and serine replaced glycine at residue 20 in resistant strain 654/ taining 5 mg/mL optochin and enriched with 3% defibrinated lysed R (figure 3). horse blood. Our finding that the optochin-susceptible and -resistant vari- Nucleotide sequence accession numbers. The nucleotide se- ants of isolate HV109 had identical ATPase c-subunit nucleotide quence data were deposited in the GenBank database under ac- sequences suggested that mutations within a different ATPase cession numbers AF334388–AF334397. subunit might also confer optochin resistance. By using primers 961 (5-TTTGGGAATATCTTTGCAGGAGAG-3) and 1791 (5-TGCGACTTGCTTGATTTGATCC-3), we cloned and se- Results quenced the remaining portion of the ATPase a-subunit, the ATPase b-subunit, and part of the ATPase x-subunit from both All optochin-resistant S. pneumoniae isolates were a-hemolytic and bile soluble, exhibited typical colony morphology, and produced pneumococcal capsular polysaccharide. Optochin MICs were 4–30-fold higher in optochin-resistant than in optochin-susceptible strains (table 1). No relationship between resistance to optochin and other antimicrobial agents tested was detected. For isolates that yielded optochin-resistant variants, both optochin-susceptible and -resistant colonies were bile soluble, demonstrated identical antimicrobial susceptibility profiles, and were of identical serotypes (table 1). The 4 optochin-resistant isolates belonged to serotypes 6B, 9V, 14, and 19F, all common serotypes in the Washington, DC, region [18]. PFGE analysis of the DNA from the optochin-resistant isolates after digestion with restriction endonuclease SmaI disclosed that the isolates were clonally unrelated. However, in isolates with optochin-resistant variants, comparison of PFGE of optochin-susceptible and -resistant colonies revealed identical profiles (figure 2). We compared the ATPase c-subunit amino acid sequences from optochin-susceptible and -resistant strains to ascertain Figure 2. Pulsed-field gel electrophoresis (PFGE) of SmaI-digested whether optochin resistance was associated with any amino acid chromosomal DNA from optochin-resistant isolates. For isolates with changes [13]. For isolates exhibiting optochin-resistantvariants, mixed populations of optochin-susceptible (S) and -resistant (R) colonies, sequence analyses were performed on both susceptible and re- PFGE was performed on both susceptible colonies and resistant variants. JID 2001;184 (1 September) Optochin Resistance in S. pneumoniae 585 Downloaded from https://academic.oup.com/jid/article/184/5/582/808236 by guest on 23 September 2021

Figure 3. Comparison of ATPase c-subunit amino acid sequences among optochin-susceptible (S) and -resistant (R) strains. For isolates with optochin-resistant variants (606a, HV109, and 654), sequencing was performed on both susceptible colonies and resistant variants. the optochin-susceptible HV109 (HV109/S) isolate and its op- the primary and, in some cases, the only method used by clinical tochin-resistant variant (HV109/R). Comparison of these se- microbiology laboratories to differentiate S. pneumoniae from quences revealed that a serine residue replaced tryptophan at other viridans streptococci [4, 21]. Strains displaying a growth position 206 in the ATPase a-subunit of the resistant variant inhibitory zone у14 mm in diameter around a 6-mm optochin (figure 4). disk are identified as S. pneumoniae. Those producing zones !14 Genetic transformation experiments confirmed that each of mm in diameter are questionable pneumococci and should be the individual amino acid changes described conferred optochin tested further to determine whether they are bile soluble [3]. When resistance. Experiments were done using the optochin-suscep- there is no inhibitory zone around the optochin disk, laboratories tible strain HV109/S as recipient and plasmid DNA encoding routinely identify isolates as viridans streptococci and do not either the ATPase a- or c-subunit from the optochin-resistant perform bile solubility testing [21]. strains ligated into pCR-Blunt vector as donor. Plasmids were Although S. pneumoniae was considered universally susceptible linearized by digestion with AspI restriction endonuclease be- to optochin for almost 30 years after the optochin susceptibility fore transformation. AspI cleaved at only one site in the cloning test was introduced, a number of isolates have been reported as vector and did not cleave the donor DNA insert. Encapsulated optochin resistant in recent years. In 1987, Kontiainen and Si- S. pneumoniae HV109/S was selected as the transformation re- vonen [10] were the first to report pneumococci that exhibited cipient because its DNA sequence differed from each of the an optochin-resistant subpopulation. A similar phenomenon was optochin-resistant strains by only 1 base in the region of in- described a year later by Phillips et al. [11]. Munõz et al. [12] terest. In addition, preliminary tests with the HV109/S strain reported that 10 (1.2%) of 814 pneumococcal isolates submit- showed that it could be reproducibly transformed by using ted to their reference center in 1987–1988 contained optochin- DNA from a streptomycin-resistant pneumococcus. Two trans- resistant variants. Three of our isolates also produced a mixed formants from each experiment were selected and tested for population of optochin-susceptible and -resistant colonies. Pre- susceptibility to optochin. Results showed identical optochin dominance of optochin-resistant variants can easily lead to mis- MICs between the transformants and the original optochin- identification of isolates as viridans streptococci. One of our iso- resistant donors. Sequence analysis of the transformants con- lates (strain 310; figure 1) and the isolate from Chicago reported firmed the expected amino acid changes. by Borek et al. [15] were uniformly resistant to optochin. Inves- tigators from Spain recently identified a gene responsible for the optochin susceptibility/resistance phenotype [13]. They found Discussion that point mutations in amino acid residues 48, 49, or 50 of the Despite the development of newer techniques (e.g., use of DNA Hϩ-ATPase c-subunit confer optochin resistance. probes and PCR) [19, 20], the optochin susceptibility test remains Hϩ-ATPase is a membrane-bound multimeric enzyme com- 586 Pikis et al. JID 2001;184 (1 September) Downloaded from https://academic.oup.com/jid/article/184/5/582/808236 by guest on 23 September 2021

Figure 4. Comparison of ATPase a-subunit amino acid sequences between optochin-susceptible strain HV109/S and its optochin-resistant variant HV109/R. Both optochin-susceptible and -resistant colonies of isolate HV109 had identical ATPase c-subunit nucleotide sequences (ﬁgure 3). plex responsible for proton translocation across plasma mem- Oligomeric complexity of ATPases varies among different or- ϩ branes. This enzyme is an F0F1 class ion transport ATPase ganisms; however, subunits involved in H translocation and found in bacteria, mitochondria, and chloroplasts. The F0 sec- ATP synthesis show remarkable amino acid homology and struc- tor of the complex comprises membrane-embedded protein sub- tural similarity. On this basis, it is believed that the enzymatic units that translocate hydrogen ions, while the F1 component mechanism is the same in all species. The ATPase from E. coli of the complex contains the catalytic subunit for ATP hydrol- is one of the best-studied models of a membrane-bound enzyme ysis and synthesis [22]. complex. Although all 3 subunits (a, b, and c) are required for

We compared sequences of ATPase a- and c-subunits from a functional F0 component, it appears that only the a- and c- various optochin-resistant and -susceptible pneumococcal subunits are directly involved in Hϩ translocation. Structural and strains and found that several single amino acid mutations seg- genetic analyses show that certain amino acid residues in the regate with optochin resistance (figures 3 and 4). Site-specific transmembrane domains of the model are essential for Hϩ trans- mutagenesis of an optochin-susceptible S. pneumoniae strain at location across the plasma membrane [22]. single amino acid residues corresponding to changes in either To understand the structural relationship between a single the a-subunit (Trp206Ser) or the c-subunit (Gly20Ser, Met23Ile, amino acid mutation and the mechanism of optochin resistance and Ala49Thr) conferred optochin resistance. These results in S. pneumoniae, we compared the sequences of the a- and c- confirm that the single amino acid change observed in our subunits of our strains with the corresponding E. coli sequences. clinical isolates is responsible for optochin resistance. Of note, We compiled table 2 and figure 5 by use of Clustal-W amino 3 of the optochin-resistant pneumococcal isolates had muta- acid alignments to the E. coli ATPase. This putative model tions at sites different than those reported from Spain [13]. shows the structural relationships between mutations that con- Moreover, this is the first report of a mutation affecting the fer optochin (or both optochin and quinine) resistance and ATPase a-subunit. Our other 2 optochin-resistant isolates had amino acid residues that are essential to Hϩ translocation. By mutations (Gly20Ser and Met23Ile) within the a-helix 1 trans- analogy to the E. coli enzyme, the putative S. pneumoniae model membrane domain of the ATPase c-subunit (figure 5). All the for the F0 portion of ATPase consists of a membrane-embedded Spanish mutations (from clinical isolates or laboratory-gener- cylinder made up of 10–12 c-subunits with 1 a-subunit posi- ated mutants after selection on plates containing optochin) had tioned within the membrane matrix on the outer surface of the mutations corresponding to the a-helix 2 domain of the ATPase cylinder. Individual c-subunits consist of 2 transmembrane a- c-subunit [13]. However, in a related study, laboratory-gener- helices joined by a short conserved cytoplasmic loop. The cyl- ated pneumococcal mutants resistant to quinine had mutations inder structure is stabilized by hydrogen bond interactions be- at the same site on a-helix 1 as our optochin-resistant clinical tween the backbone carbonyl of valine 15 in a-helix 1 and the isolates [26]. These quinine-resistant mutants, which also ex- carboxyl side chain of glutamate 52 in a-helix 2 of an adjacent hibited cross-resistance to optochin, were obtained by spreading c-subunit (and possibly between valine 48 in a-helix 2 and glu- R6 cells on plates containing quinine. An optochin-resistant tamate 19 in a-helix 1; figure 5). The protein cylinder acts as pneumococcal clinical isolate with a mutation (Gly14Ser) cor- a rotor embedded in the membrane. The proton pathway responding to the transmembrane a-helix 1 was described re- through the membrane is thought to be formed by an opening cently in France [27]. of the interface between adjacent c-subunits by swiveling of JID 2001;184 (1 September) Optochin Resistance in S. pneumoniae 587 Downloaded from https://academic.oup.com/jid/article/184/5/582/808236 by guest on 23 September 2021

ϩ Figure 5. Putative model for Streptococcus pneumoniae F0 sector of H translocation domain of ATPase. Hairpin structure of 1 c-subunit with transmembrane a-helices 1 and 2 is shown as are transmembrane a-helices 4 and 5 of a-subunit. Amino acid sequence(s) of S. pneumoniae ATPase a- and c-subunits are positioned in the model according to their function and equivalent position in the Escherichia coli model. Yellow circles, amino acid residues identical to those of the E. coli sequence; orange circles, residues that are similar in structure; open circles, residues unique to S. pneumoniae enzyme. Residues that are essential to stabilizing adjacent c-subunits in the cylinder structure (V15 with E52 and possibly V48 with E19) or essential to Hϩ translocation across the membrane (E52 in c-subunit and R170, E179, and S210 in a-subunit) are indicated by residue no. in sequence. Residues within the cytoplasmic loop of the c-subunit and E160 in the a-subunit are highly conserved among different species. Amino acid mutations that are responsible for optochin resistance are marked with red and show residue no., followed by amino acid mutation that confers resistance (G14S, G20S, M23I, V48F, A49T, and F50L in c-subunit and W206S in a-subunit). Figure is based on the work of Miller et al. [23], Hatch et al. [24], Fillingame et al. [25], and de la Campa et al. [26]. helix 1, thus allowing a hydrogen from the protonated gluta- amino acids thought to be essential to hydrogen transport. Al- mate 52 carboxyl side chain on helix 2 to transfer to the a- though the molecular mechanism of exactly how optochin in- subunit on the outer surface of the cylinder [33]. teracts with the F0 portion of ATPase is still unresolved, we In our putative model, all amino acid mutations that confer believe that in the presence of optochin the proton-translocating optochin resistance are located within those regions of helices subunits of ATPase are perturbed in such a way that the Hϩ that transverse the membrane. No mutations were observed in pump fails and the cell dies. In optochin-resistant S. pneumon- 588 Pikis et al. JID 2001;184 (1 September)

Table 2. Streptococcus pneumoniae/Escherichia coli Hϩ-ATPase subunit model with equivalent amino acids. S. pneumoniae E. coli Function identiﬁed with residue in ATPase-positive model References c-Subunit amino acids G14S G18 Confers optochin resistance in clinical S. pneumoniae isolates [27] V15 A24 Backbone carbonyl H bond with carboxyl of D61 in E. coli; [22] mutation also confers dicycloxenyl carbodiimide (DCCD) resistance in E. coli E19 I28 Mutation confers DCCD resistance in E. coli [22] G20S G29 Confers optochin resistance in clinical S. pneumoniae isolates Present study Quinine-induced resistance in S. pneumoniae results in mutation [26] at G20

M23I G32 Confers optochin resistance in clinical S. pneumoniae isolates Present study Downloaded from https://academic.oup.com/jid/article/184/5/582/808236 by guest on 23 September 2021 Quinine-induced resistance in S. pneumoniae results in same [26] amino acid change R32 R41 Essential residue in the cytoplasmic loop in E. coli [28] Q33 Q42 Reacts with E31 of e-subunit and Y205 of g-subunit in E. coli [28] V48F M57 Confers optochin resistance in clinical S. pneumoniae isolates; [13] optochin-induced resistance in S. pneumoniae results in mutation at V48 A49T G58 Confers optochin resistance in clinical S. pneumoniae isolates Present study, [13] Optochin-induced resistance in S. pneumoniae results in same [13] mutation F50L L59 Confers optochin resistance in clinical S. pneumoniae isolates [13] E52 D61 Carboxyl H bond with backbone carbonyl of A24 in E. coli; [29, 30] H ion binding site in transport mechanism in E. coli Reaction with DCCD blocks H ion translocation and ATPase [31, 32] activity in E. coli E19 A24D Aspartyl-interchange mutation retains ATPase function in E. coli [23] A49 D61A Aspartyl-interchange mutation retains ATPase function in E. coli [23] a-Subunit amino acids R170 R210 Critical for conducting H ions in transport mechanism in E. [24, 29] coli; receives protons for transfer to D61 in c-subunit E179 E219 Critical for conducting H ions in transport mechanism in E. coli [29] W206S W241 Confers optochin resistance in clinical S. pneumoniae isolates Present study S210 H245 Critical for conducting H ions in transport mechanism in E. coli [29]

iae, amino acid changes within the a- or c-subunits prevent a given environment provides selective pressure that results in optochin from disrupting the Hϩ transport path. the enrichment of resistant strains. Although optochin is used The origin of optochin resistance in S. pneumoniae is unclear. only as a diagnostic tool in clinical microbiology laboratories, S. pneumoniae is a naturally transformable microorganism, and, similar compounds such as quinine and mefloquine are widely in many cases, resistance to certain antibiotics has been ascribed used for treatment and prophylaxis against malaria. Each year to acquisition of either free DNA from other species followed several thousand American or foreign travelers arrive in the by DNA recombination or by the transfer of conjugative trans- Washington, DC, metropolitan area from malaria-endemic ar- posons encoding antimicrobial resistance genes [34]. Resistance eas, and imported malaria is not uncommon [37]. Even though due to single point mutations is less common but has been none of our patients with optochin-resistant S. pneumoniae was demonstrated for high-level trimethoprim resistance [35] and treated for malaria or had a travel history to a malaria-endemic low-level fluoroquinolone resistance [36]. Our findings indicate area, possible acquisition of these unique pneumococcal strains that optochin resistance in S. pneumoniae is a result of point from persons who did seems plausible and cannot be ruled out. mutations in either the ATPase a- or c-subunit. This hypothesis Further epidemiological studies to investigate the incidence of is strongly supported by the findings from the 3 isolates with optochin resistance in S. pneumoniae in malaria-endemic areas optochin-resistant variants. In those mixed isolates, the iden- are needed to clarify a possible role of quinine and other similar tical PFGE profile, serotype, and antibiogram, together with analogues in the evolution of optochin resistance. sequence analysis and transformation experiments, indicate Misidentification of optochin-resistant S. pneumoniae as a that resistant variants originated from susceptible cells by single viridans streptococcus can have significant implications for the point mutations. treatment and outcome of patients. We recommend that at a It is intriguing that the mutations at positions 20 and 23 of minimum invasive a-hemolytic streptococci resistant to opto- the c-subunit of our clinical isolates are in the same position chin be checked for lack of bile solubility before being identified as the mutations observed in laboratory-derived pneumococcal as viridans streptococci. Even though bile-insoluble pneumo- mutants resistant to quinine. The presence of antibacterials in coccal isolates have been reported [21], the addition of this latter JID 2001;184 (1 September) Optochin Resistance in S. pneumoniae 589 test should minimize the possibility of misidentification. A com- resistant Enterococcus faecium colonization in children. J Clin Microbiol mercial DNA probe test is of higher sensitivity and specificity 1999;37:413–6. than standard tests [19] but may be too expensive for routine 17. Lacks S. Integration efficiency and genetic recombination in pneumococcal transformation. Genetics 1966;53:207–35. use. However, it should be considered for evaluation of isolates 18. Pikis A, Akram S, Donkersloot JA, Campos JM, Rodriguez WJ. Penicillin- with a typical colony morphology for S. pneumoniae that yield resistant pneumococci from pediatric patients in the Washington, DC, questionable results by other methods. area. Arch Pediatr Adolesc Med 1995;149:30–5. 19. Denys GA, Carey RB. Identification of Streptococcus pneumoniae with a Acknowledgments DNA probe. J Clin Microbiol 1992;30:2725–7. 20. Zhang Y, Isaacman DJ, Wadowsky RM, Rydquist-White J, Post JC, Ehrlich

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