Diversity of Mutations in the atpC Gene Coding for the c Subunit of F₀F₁ ATPase in Clinical Isolates of Optochin-Resistant Streptococcus pneumoniae from Brazil

Abstract

We report the characteristics of four optochin-resistant (Opt^r) Streptococcus pneumoniae isolates from Brazil. All four Opt^r isolates presented mutations in the nucleotide sequence coding for the c subunit of F₀F₁ ATPase. Two isolates showed mutations in codons 23 (leading to the deduced amino acid substitution isoleucine instead of alanine) and 49 (serine instead of alanine, a novel type of mutation detected at this position), respectively. Two additional novel mutations, both located in codon 45, were detected in the other two isolates, corresponding to leucine or valine (instead of phenylalanine). The data indicate that three previously unrecognized alterations were detected in the atpC gene of S. pneumoniae and that Opt resistance among Brazilian pneumococcal isolates is not related to a specific pneumococcal serotype, antimicrobial-resistance profile, or clonal group.

Streptococcus pneumoniae is one of the most important human pathogens, remaining as a major cause of community-acquired infections, such as pneumonia, bacteremia, meningitis, otitis media, and sinusitis (3). Because of the increasing frequency of antimicrobial resistance, accurate identification and antimicrobial susceptibility testing are essential for correct diagnosis and treatment of patients. Determination of phenotypic characteristics is conventionally used in diagnostic laboratories for the identification of S. pneumoniae, including colony morphology on blood agar plates, optochin (Opt) susceptibility, bile solubility, and reactivity with type-specific antisera for detection of capsular polysaccharide antigen (1, 11). Several commercial systems and rapid kits are also available. More recently, a variety of molecular methods, including a DNA probe directed to a section of rRNA (8), PCR assays for detection of genes encoding diverse virulence factors (4, 12, 14), and DNA-DNA reassociation (1), have been applied to identify pneumococcal isolates. Despite the development of newer methods, most routine laboratories still rely on the results of Opt susceptibility testing as the primary or even the only test for the presumptive identification of pneumococci. Occasionally, however, isolates of S. pneumoniae exhibiting an optochin-resistant (Opt^r) phenotype have been reported (2, 6, 7, 10, 13, 15). The occurrence of such a phenotypic variant is a potential cause of problems in the precise characterization of this agent, leading to misidentification. The Opt^r phenotype is attributed to mutations in the atpC gene that codes for the molecular target of Opt, the transmembrane F₀F₁ ATPase, involved in proton transportation in the respiratory chain (9, 13). Alterations in α-helix 1, corresponding to codons 14, 20, and 23 (6, 13), and in α-helix 2, corresponding to codons 48, 49, and 50 (7, 13) of the c subunit of the F₀ complex of the molecule, have already been described in clinical isolates of S. pneumoniae. A mutation located in the a subunit of F₀F₁ ATPase was also described (13). To date, reports of the occurrence of Opt^r pneumococci are still sporadic, and only a few isolates with the Opt^r phenotype have had their respective mutations described.

The purpose of this communication is to report the occurrence and characterization of Opt^r S. pneumoniae clinical isolates from Brazil, illustrating the global diversity among isolates from different geographic areas.

Four S. pneumoniae isolates (Sp 910, Sp 913, Sp 917, and Sp 1008) presenting the Opt^r phenotype were investigated. Two of them (Sp 910 and Sp 913) were recovered from lower respiratory tract specimens, one (Sp 917) was isolated from blood, and the other (Sp 1008) was isolated from ocular secretion. These isolates were detected among a collection of about 470 pneumococci obtained in the period 1995 to 1999 during studies of the antimicrobial susceptibility, serotype distribution, and molecular epidemiology of pneumococcal isolates recovered from individuals living in Porto Alegre City, Rio Grande do Sul State, located in the southern region of Brazil. Three of them (Sp 910, Sp 913, and Sp 917) were isolated in 1995, and one (Sp 1008) was isolated in 1996. The isolates were initially subjected to conventional identification tests, including observation of colony characteristics on blood agar plates, cellular characteristics as observed after Gram staining, optochin susceptibility, and bile solubility, according to previously described methods (1, 11). Serotyping was based on capsular swelling (the Quellung reaction) with type-specific pneumococcal antisera (Centers for Disease Control and Prevention, Atlanta, GA), using the Danish system of nomenclature. Susceptibility to antimicrobial agents (ceftriaxone, chloramphenicol, erythromycin, penicillin, tetracycline, and vancomycin) was evaluated by an agar dilution method, based on the Clinical and Laboratory Standards Institute (5) guidelines. The presence of the virulence genes ply (14), psaA (12), and lytA (4) was detected by PCR procedures. A commercial DNA probe (AccuProbe S. pneumoniae culture identification test; Gen-Probe, San Diego, CA) was used, following the manufacturer's instructions. Chromosomal DNA macrorestriction profiles were obtained by treatment with SmaI, followed by pulsed-field gel electrophoresis (PFGE) (11). In order to identify mutations responsible for the Opt^r phenotype, primers 663 (5′-TCGAAAAGTGGATCAACAACTATCC-3′) and 1016 (5′-TGGGAAAGAAGAAGTAACAAACTCG-3′) were used to amplify a 930-bp fragment coding for the c subunit of the F₀F₁ ATPase gene (9). After purification of the amplicons, sequencing was carried out by the Big Dye Terminator method v3.1 (Perkin-Elmer Applied Biosystems, Foster City, CA), using primer 663 in an automated system (ABI Prism 310 Genetic Analyzer; P-E Applied Biosystems) according to the manufacturer's recommendations. An Opt-susceptible (Opt^s) reference strain (S. pneumoniae R6) was also included in the sequencing experiments for comparative purposes.

No zone of inhibition was observed around optochin disks when the four Opt^r isolates were tested. The observations were consistent when Opt susceptibility tests were incubated under two different conditions, ambient atmosphere and CO₂. Except for resistance to Opt, the four isolates exhibited typical phenotypic and genetic profiles for identification as S. pneumoniae: they all were bile soluble; presented a capsular antigen; possessed the ply, lytA, and psaA genes; and hybridized with the pneumococcal genetic probe. A comparison with the wild-type atpC gene sequence of the Opt^s strain R6, however, revealed that all four isolates with the Opt^r phenotype presented mutations located in the gene coding for the c subunit of F₀F₁ ATPase (Table 1). Isolates Sp 910 and Sp 913 presented changes in codons 23 and 49, respectively. Mutations in these two codons have been previously described, although the nucleotide change observed in isolate Sp 913 was unique, comprising a G-to-T transition at position 145 of the atpC gene sequence obtained in the present study and corresponding to position 357 of the published atpCAB gene cluster sequence (GenBank Z26851). This transition produces an alanine-to-serine amino acid substitution in a region corresponding to transmembrane α-helix 1 of the AtpC protein, which differs from two previous reports (7, 13) describing an alanine-to-threonine substitution. Additionally, two novel mutations, both located in codon 45, were observed in two other isolates (Sp 917 and Sp 1008). The nucleotide changes detected corresponded to two different deduced amino acid substitutions: leucine (instead of phenylalanine) in isolate Sp 917 and valine in isolate Sp 1008. The four isolates belonged to different serotypes, as shown in Table 1. Analysis of PFGE profiles indicated that the isolates were not genetically related to each other (data not shown). The isolates were susceptible to most of the antimicrobial agents tested, except for Sp 910, which was resistant to chloramphenicol and sulfamethoxazole-trimethoprim, and Sp 1008, which was resistant to tetracycline.

TABLE 1.

General characteristics and mutations in the atpC gene coding for the c subunit of F₀ F₁ ATPase in Opt^r S. pneumoniae isolates

Isolate	Serotype or serogroup	Location of mutationa	Locality of isolation	Resistance to antimicrobial agentsb	Genetic relatedness among strains in the study	Reference
MJ9559	19	49 (Ala→Thr)	Spain	NP/NRc	NP/NR	7
MJ455	6	48 (Val→Phe)	Spain	NP/NR
MJ1966	14	50 (Phe→Leu)	Spain	NP/NR
MJ4133	NTd	48 (Val→Phe)	Spain	NP/NR
P21	23	14 (Gly→Ser)	France	NP/NR	NAe	6
310	9V	23 (Met→Ile)	United States	None	Unrelated	13
660	14	49 (Ala→Thr)	United States	None	PFGE profiles
654	19F	20 (Gly→Ser)	United States	CHL, PEN
Sp 910	6A	23 (Met→Ile)	Brazil	CHL, SXT	Unrelated	Present
Sp 913	23F	49 (Ala→Ser)	Brazil	None	PFGE profiles	study
Sp 917	6B	45 (Phe→Leu)	Brazil	None
Sp 1008	10A	45 (Phe→Val)	Brazil	TET

^aCodon no. (amino acid found in Opt^s S. pneumoniae R6 reference strain → deduced amino acid substitution found in the isolate being referred).

^bCHL, chloramphenicol; PEN, penicillin; SXT, sulfamethoxazole-trimethoprim; TET, tetracycline.

^cNP/NR, not performed or not reported.

^dNT, nontypable.

^eNA, not applicable.

This is the first report of the characterization of Opt^r S. pneumoniae occurring in Brazil. Furthermore, the present communication contains a description of three previously unrecognized alterations occurring in the atpC gene of S. pneumoniae. Such alterations may be considered putative contributors to the Opt^r phenotype, since the results of genetic transformation experiments have demonstrated that point mutations previously detected in the atpC gene were associated with Opt^r in pneumococcal isolates (9, 13). The overall data indicate that the occurrence of Opt resistance among Brazilian pneumococcal isolates is not related to a specific pneumococcal serotype, antimicrobial-resistance profile, or clonal group. Such a finding has also been observed among isolates from the United States (13). When taken in conjunction with the results of other studies, this investigation additionally illustrates the diverse nature of putative molecular mechanisms of Opt resistance in S. pneumoniae and contributes to the global knowledge about the occurrence and diversity of Opt^r pneumococcci.

The prevalence of Opt resistance among S. pneumoniae remains unknown, and it is probably underestimated, as many clinical laboratories still depend on Opt susceptibility testing for screening and identification of this microorganism and therefore may overlook or misidentify S. pneumoniae isolates with the Opt^r phenotype. Clinicians and microbiologists should be aware of the existence of the Opt^r pneumococcal isolates circulating in various areas and consider them a potential cause of life-threatening infections. Since misidentification of Opt^r S. pneumoniae as viridans streptococci may have significant implications for the management of patients, routine use of at least one additional test, such as the bile solubility test, should be adopted to accurately identify S. pneumoniae. As more attention is dedicated to properly detecting and characterizing Opt^r pneumococcal isolates in the clinical setting, more information will became available on the occurrence and diversity of these atypical variants and their roles as agents of infections.

Nucleotide sequence accession numbers.

The sequences of the atpC gene reported here have been deposited in the GenBank database under the following accession numbers: EF464066 (isolate Sp 910), EF464067 (isolate Sp 913), EF464068 (isolate Sp 917), and EF464069 (isolate Sp 1008).