Multidrug Resistance in Non-PCV13 Serotypes of Streptococcus pneumoniae in Northern Japan,2014

Pneumococcal strains

In our previous study, the distribution of serotypes and macrolide resistance genotypes of 2057 noninvasive isolates of S. pneumoniae from April 2013 to November 2014 was described. ¹⁴ In this study, in a PCV13 routine immunization period between July and December 2014, a total of 293 nonduplicate isolates from noninvasive infections or colonization (i.e., isolates from nonsterile specimens) were derived from outpatients of children (<16 years of age) who visited various hospitals and clinics throughout Hokkaido, the northern main island of Japan. Among them, all the isolates of non-PCV13 serotypes (231 isolates) were analyzed in the present study. The NVT isolates were assigned to 12 serotypes, including the common types 6C (16.9%), 15A (16%), 23A (13.9%), 15C (11.7%), 10A (11.3%), and 35B (9.1%), which accounted for 78.9% of all the NVT isolates. All isolates were stored in Microbank (Pro-Lab Diagnostics, Richmond Hill, Canada) at −80°C until analysis.

Serotyping and discrimination of serotypes 15A from 15F by PCR

In our previous studies, multiplex polymerase chain reactions (M-PCRs) were used to determine serotypes, including 15A, as described by Pai et al. in 2006. ¹⁵ However, thereafter, serotype 15A determined by the method ¹⁵ was revealed to contain 15F as well as 15A, which was alerted by the CDC website (www.cdc.gov/streplab/pcr.html). Between serotypes 15A and 15F, there is a difference in length with approximately 130 bp sequence in wciZ gene (GenBank accession numbers CR931663 for serotype 15A and CR931666 for serotype 15F). ¹⁶ In the present study, serotypes 15A and 15F were discriminated by PCR using newly designed primers 15bc-n1 (5′-TGGAGAGAAGGAATATGCAG-3′) and 15bc-n2 (5′-CTATTCCAAAAAATGGTATTCCCA-3′) producing different sizes of products (165 bp and approximately 290 bp for 15A and 15F, respectively, based on the earlier reference sequences). Using this method, all the isolates previously assigned to serotype 15A were confirmed to have a genetic trait of 15A.

Antimicrobial susceptibility testing

Susceptibilities to penicillins [PEN, ampicillin (AMP)], ampicillin–sulbactam (SAM), macrolides [erythromycin (ERY), clarithromycin (CLR), azithromycin (AZM)], clindamycin (CLI), cephalosporins [cefepime (FEP), cefuroxime (CXM), ceftriaxone (CRO)], carbapenems [imipenem (IPM), meropenem (MEM)], quinolones [levofloxacin (LVX), moxifloxacin (MXF), gatifloxacin (GAT)], vancomycin (VAN), tetracycline (TET), trimethoprim–sulfamethoxazole (SXT) were measured by the broth microdilution method using “Dry Plate, ‘Eiken’ HW04 (Eiken Chemical Co., Tokyo, Japan)” for all isolates. By this method, the minimal inhibitory concentration (MIC) was measured within a limited range of concentration. S. pneumoniae ATCC49619 was used as a quality control strain for the testing. Susceptibility class (susceptible [S], intermediate [I], or resistant [R]) was determined according to the Clinical and Laboratory Standards Institute (CLSI) M100-S24, ¹⁷ in which breakpoints for oral administration [S, I, R (μg/ml): ≤0.06, 0.12–1, ≥2, respectively] were applied to PEN. Because breakpoints for AMP and SAM are not provided by the CLSI, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) Clinical Breakpoint Table v. 6.0, valid from January 1, 2016, ¹⁸ was used to interpret susceptibilities. Nonsusceptibility to PEN (MIC, ≥0.12 μg/ml), combined with resistance to two or more non-β-lactam antimicrobial classes, were defined as MDR.^2,6

Detection of resistance genes and assignment of PBP genotypes

The prevalence of macrolide resistance genes [erm(B) and mef(A/E)] for S. pneumoniae isolates had been investigated in our previous study. ¹⁴ In the present study, a tetracycline resistance gene tet(M) was detected by PCR as described elsewhere. ¹⁹ The presence of the three unaltered pbp genes (pbp1a, pbp2x, and pbp2b) was examined by M-PCR as described previously.^20,21 According to the previously published scheme,^20,21 genotype of penicillin resistance (PBP genotypes) was represented as gPRSP (three pbp gene alterations) and gPISP (one or two pbp gene alterations). gPISP was expressed with the altered PBP gene(s) in a parenthesis, for example, gPISP (pbp2b).

MLST and sequence analysis of pbp1a, pbp2x, and pbp2b genes

To investigate the molecular characteristics of PNSP, we selected the representative 20 isolates of dominant serotypes, including all PRSP isolates and PISP with different resistance patterns and/or MDR [PRSP; 15A (eight isolates) and PISP; 15A, 35B, 23A, 6C (three isolates each)] for analysis by sequence types (STs) through the MLST scheme and sequences of the transpeptidase (TP) region of PBP1a, PBP2x, and PBP2b. The MLST protocol with primers for amplification and sequencing described in the S. pneumoniae MLST website (http://pubmlst.org/) was used, and the STs were also assigned at the database. The obtained STs of isolates, together with serotypes, were compared with PMEN international clones (www.sph.emory.edu/PMEN). Partial pbp1a, pbp2b, and pbp2x gene sequences encoding TP-domains (1119, 1053, and 1398 bp, respectively) were determined by PCR and direct sequencing as previously described.^22,23 The PCR products were sequenced on an automated sequencer (Applied Biosystems PRISM 3130) using the Applied Biosystems Big Dye^® Terminator v1.1 Cycle Sequencing Kit. The obtained sequence data were analyzed with BLAST (http://blast.ncbi.nlm.nih.gov/Blast), and alignment of sequences was performed by ClustalW program (http://clustalw.ddbj.nig.ac.jp) and Molecular Evolutionary Genetic Analysis software (MEGA5.05), comparing with those of reference strain R6 (GenBank accession numbers M90527 for pbp1a, X16367 for pbp2x, and X16022 for pbp2b).

Statistical analysis

Statistical analysis was performed by SPSS 19.0 (IBM). A two-tailed chi-square test or Fisher's accurate probability methods (for small group sizes) were used to compare proportions of antimicrobial susceptibility in each serotype. A p < 0.05 was considered statistically significant.

Nucleotide sequence accession numbers

The pbp1a, pbp2x, and pbp2b gene sequences containing TP-domain-encoding regions of 13 PNSP isolates were deposited to GenBank under accession nos. KU749446–KU749484.

Antimicrobial susceptibility

The percentages of intermediate resistance (I) and resistance (R) of all isolates with non-PCV13 serotypes are summarized in Table 1. All isolates were susceptible to LVX, MXF, GAT, and VCM (data not shown). PISP and PRSP were detected in 39.4% and 3.5% of the isolates, respectively. All the serotype 15A isolates were nonsusceptible to PEN (PRSP, 21.6%; PISP, 78.4%), and PISP was found in serotypes 23A (96.9%), 6C (41.0%), 35B (33.3%), 15C (18.5%), 15B (8.3%), 11A (6.3%), and 10A (3.8%).

A high rate of nonsusceptible isolates to macrolides (ERY, CLR, and AZM), TET, and CLI (≥92.7%, 92.6%, and 75.3%, respectively) was observed. Of note, serotypes 15A, 23A, 15C, and 35B were 100% resistant to macrolides and TET. Among serotypes 15A and 35B, considerable isolates showed intermediate susceptibility to IPM (51.4% and 19.0%, respectively), MEM (32.4% and 23.8%, respectively), and CXM (48.6% and 23.8%, respectively). All the PNSP of serotype 35B were nonsusceptible to CXM. In addition, all the serotypes 15A and 35B were nonsusceptible to SXT. Overall, MDR was observed in 42.0% (97/231) of NVT isolates, containing dominant types 15A (n = 37) and 23A (n = 31), followed by 6C (n = 14), 35B (n = 7), and 15C (n = 5).

MLST and resistance gene genotypes in PNSP strains

Among the PNSP isolates of serotypes 15A, 35B, 23A, and 6C, representative 20 isolates were selected for further genetic analysis. In terms of MLST, 90% (18/20) of these isolates revealed to belong to PMEN clones or their single-locus variants (SLVs) (Table 2). All the serotype 15A isolates were classified into ST63, that is, PMEN clone Sweden^15A-25 (international clone), and serotype 35B isolates were assigned to ST558, which is an SLV in ddl gene of Utah^35B-24 clone ST377. Among the serotype 23A isolates, one isolate was assigned to ST338 (Colombia^23F-26), while two isolates to ST5242, an SLV of ST338. Among serotype 6C, one isolate was assigned to ST242 (Taiwan^23F-15), while remaining two isolates to ST5832.

PBP gene genotypes and genes responsible for macrolides and tetracycline resistance are shown in Table 2. Serotypes 15A, 6C, and 35B with PEN-MIC of 0.5–2 μg/ml were assigned to gPRSP, while serotype 23A isolates with PEN-MIC of 0.5–1 μg/ml were assigned to gPISP (pbp2x+2b and pbp2b). Remaining serotypes 15A and 6C isolates (PEN-MIC, 0.12 μg/ml) were typed as gPISP (pbp1a+2b and pbp2x+2b, respectively). All these isolates possessed tetracycline resistance determinant tet(M), and either macrolide resistance gene erm(B) or mef(A/E). Most of isolates with erm(B) showed high-level resistance to ERY (MIC, ≥128 μg/ml).

Sequence analysis of pbp1a, pbp2x, and pbp2b genes

The representative 20 isolates were further genetically analyzed for the TP-regions encoded by pbp1a, pbp2x, and pbp2b (Figs. 1–3). In PBP1a (Fig. 1), a mutation T₃₇₁S in the ₃₇₀STMK₃₇₃ motif was found in all the serotype 15A PRSP isolates and PISP isolates with serotypes 15A, 6C/ST242, and 35B. On the contrary, an amino acid substitution of P₄₃₂T in ₄₂₈SRNVP₄₃₂ motif was observed in seven of eight isolates of PRSP and 25% of PISP isolates. Four consecutive substitutions, TSQY to NTGY at positions 574–577, were also found in all the PRSP isolates and 75% of PISP isolates. Amino acid substitutions in TP regions of PBP2x were T₃₃₈A (17/20) and M₃₃₉F (8/20) in the ₃₃₇STMK₃₄₀ motif, H394L (3/20) in the ₃₉₄HSSN₃₉₇ motif, and L₅₄₆V (17/20) in the ₅₄₆LKSGT₅₅₀ motif (Fig. 2). In addition, a mutation of STMK_337–340SAFK was found in eight PNSP (PEN; MIC of ≥0.12 μg/ml) isolates with nonsusceptibility to cephems (CXM; MIC of ≥8 μg/ml, FEP and CRO; MIC of ≥2 μg/ml), except for two isolates (Table 2 and Fig. 2). In contrast, a mutation of H₃₉₄L was observed only in three isolates in serotypes 6C and 23A, which had no mutations in other two motifs. In the PBP2b (Fig. 3), all isolates possessed only one mutation T₄₄₅A in ₄₄₂SSNT₄₄₅ motif. Other motifs had no amino acid substitutions in any isolates.

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FIG. 1.

Alignment of the amino acid sequence (amino acid position) of the PBP1a transpeptidase domain region of PNSP isolates. Amino acids were compared with those of the penicillin-susceptible Streptococcus pneumoniae R6 strain in boldface. Amino acids identical to those of R6 reference strain (GenBank accession no. M90527) are represented by dots. The penicillin-binding motifs are shown in light gray. Substitutions at positions 371, 432, and 574–577 in the motif are shown in dark gray. MIC against PEN (μg/ml); ≤0.06, 0.12–1, ≥2, respectively. MIC, minimum inhibitory concentration; PEN, penicillin; PNSP, penicillin-nonsusceptible pneumococci. I, intermediate; R, resistant; S, susceptible.

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FIG. 2.

Alignment of the amino acid sequence (amino acid position) of the PBP2x transpeptidase domain region of PNSP isolates. Amino acids were compared with those of the penicillin-susceptible S. pneumoniae R6 strain in boldface. Amino acids identical to those of R6 reference strain (GenBank accession no. X16367) are represented by dots. The penicillin-binding motifs are shown in light gray. Substitutions at positions 338, 339, 394, and 546 in the motif are shown in dark gray. S, I, R: MIC against PEN (μg/ml); ≤0.06, 0.12–1, ≥2, respectively.

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FIG. 3.

Alignment of the amino acid sequence (amino acid position) of the PBP2b transpeptidase domain region of PNSP isolates. Amino acids were compared with those of the penicillin-susceptible S. pneumoniae R6 strain in boldface. Amino acids identical to those of R6 reference strain (GenBank accession no. X16022) are represented by dots. The penicillin-binding motifs are shown in light gray. Substitutions at position 445 in the motif are shown in dark gray. S, I, R: MIC against PEN (μg/ml); ≤0.06, 0.12–1, ≥2, respectively.