Directly Repeated Insertion of 9-Nucleotide Sequence Detected in ...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1996, p. 1257–1259 0066-4804/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 40, No. 5

Directly Repeated Insertion of 9-Nucleotide Sequence Detected in Penicillin-Binding Protein 2B Gene of Penicillin-Resistant Streptococcus pneumoniae AKIO YAMANE,1* HIROMI NAKANO,1 YASUKO ASAHI,2 KIMIKO UBUKATA,2 2 AND MASATOSHI KONNO Institute for Biotechnology Research, Wakunaga Pharmaceutical Co., Ltd., 1624 Shimokotachi, Koda-cho, Takata-gun, Hiroshima 739-11,1 and Department of Clinical Pathology, School of Medicine, Teikyo University, Itabashi-ku, Tokyo 173,2 Japan Received 16 August 1995/Returned for modification 8 November 1995/Accepted 13 February 1996

We investigated the molecular mechanism of 50 penicillin-resistant Streptococcus pneumoniae strains (penicillin: MIC, ^0.125 mg/ml) having neither class A nor class B mutations in the penicillin-binding protein 2B gene (pbp2b). An analysis of the nucleotide sequences of the pbp2b genes from seven strains revealed an unique direct repeat of 9 nucleotides (TGGTATACT) between active-site serine (residue 385) and Ser-X-Asn (residues 442 to 444) motifs. The same insertion was detected in 13 strains. 59 end with biotin and antisense primer PBP2 (59-CAATTAG CTTAGCAATAGGTGTTGG; nucleotides 2,292 to 2,316). Amplified DNA for the template was purified with streptavidin-coated magnetic beads for the sequencing reaction (8). Primers P2B6 and P2B7 labeled with fluorescein were used as sequencing primers. Sequences of about 420 bp of both strands from all seven strains were determined with Sequenase, version 2.0, and an automated fluorescent DNA sequencer (DSQ-1; Shimadzu Co., Kyoto, Japan). The nucleotide sequences of the seven strains were aligned together with penicillin-susceptible S. pneumoniae (PSSP) R6 (7), class A PRSP 53139/72 (5), class B PRSP DN87/577 (5), and penicillin-resistant Streptococcus mitis 7026 (4) (Fig. 1). No typical mutations found in class A or class B PRSP strains were detected in these strains. It was noteworthy that three strains (strains KK3, NA9, and UB30) possessed an unique set of 9 nucleotides (TGGTATACT) that are repetitions of the immediately adjacent sequences at the insertion site. This direct repeat was present near the region corresponding to characteristic class A and class B mutations. Sporadic mutations detected in strains KY7, KK8, and KS13 were also confirmed in the strains KK3, NA9, and UB30. The sequence of strain OH01 differed slightly from those of susceptible strains. Residues of 115 amino acids from seven strains were compared with the amino acids from PSSP R6, class A and class B PRSP strains, and S. mitis (Fig. 2). Three conserved amino acid motifs located on the transpeptidase domain, the Ser-X-X-Lys tetrad (residues 385 to 388), the Ser-X-Asn triad (residues 442 to 444), and Glu (residue 475), are boxed. Three amino acid residues (Trp-Tyr-Thr) corresponded to the 9 direct repeat nucleotide sequences located between the Ser-X-X-Lys and the Ser-X-Asn motifs. Two substitutions, of the Thr residue at position 445 (Thr-445) to a Ser or an Ala residue and of the Glu-475 residue to a Gly residue, appeared to be common to six strains, but not to strain OH01, and a substitution of Thr488 to Ala or Ser was found in five strains. To detect the 9-bp insertion in ‘‘unknown’’ strains, PCR was conducted with primers P2BR5 (59-TAAGCCTGAGTATAC CAAGT; nucleotides 1,505 to 1,515 plus 9 nucleotides) and P2B6, which amplify them specifically. This unique sequence was detected in 13 of 50 strains, and the MICs for all strains ranged from 0.125 to 2 mg/ml for penicillin and 0.063 to 1

Penicillin-resistant Streptococcus pneumoniae (PRSP) strains pose a serious problem worldwide (1), with many studies of the resistance mechanism showing that alterations of three penicillin-binding proteins (PBPs), PBPs 1A, 2X, and 2B, are involved in the high-level resistance to penicillin (2). Of the genes encoding each PBP, PBP 2B gene (pbp2b) mutations in PRSP have been classified into class A, class B, and other mutations (4, 5, 10). We have already used PCR to investigate the correlation between the MICs of penicillin and the presence of mutations in the pbp2b gene (12). Of 441 clinical PRSP isolates (penicillin MICs, ^0.125 mg/ml) collected from institutions throughout Japan, the class A mutation was detected in only eight strains (1.8%), but the class B mutation was detected in 310 strains (70.3%). The remaining 123 strains (27.9%) were found to have neither class A nor class B mutations. These strains were classified as ‘‘unknown’’ because of the possibility that they possess a mutation(s) in regions different from those of typical class A or class B mutations. On the basis of these results, we determined the nucleotide sequences of the pbp2b genes from seven strains selected at random from among the 123 non-class A and non-class B strains. Genomic DNAs were prepared by a previously described method (11). The bacterial colony was treated with a proteinase K solution, and the mixture was incubated at 958C for 10 min to inactivate enzymes. One microliter of this preparation was used as the PCR template. In the first step, a 1.5-kb DNA fragment, including a coding region of the transpeptidase domain surrounding the active-site serine residue of the pbp2b gene, was amplified by previously described methods (6). The second PCR was conducted with two pairs of primers (4): (i) an upstream pair, sense primer PBP1 (59-GATCCTCTAAAT GATTCTCAGGTG; nucleotides 812 to 835) and antisense primer P2B7 (59-CGAGGAGCCACACGAACACC; nucleotides 1,844 to 1,863) labeled at the 59 ends with biotin, and (ii) a downstream pair, sense primer P2B6 (59-ATTCCTTGG GAACGGTAACC; nucleotides 1,347 to 1,366) labeled at the * Corresponding author. Mailing address: Institute for Biotechnology Research, Wakunaga Pharmaceutical Co., Ltd., 1624 Shimokotachi, Koda-cho, Takata-gun, Hiroshima 739-11, Japan. Phone: 81-82645-2331. Fax: 81-826-45-4351. 1257

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ANTIMICROB. AGENTS CHEMOTHER.

FIG. 1. Nucleotide sequences of part of the pbp2b genes from PSSP and PRSP strains. The sequence of PSSP R6 is shown in line a (numbering is based on data in reference 6). Only those nucleotides that differ from the R6 sequence are shown. Line b, PRSP 53139/72 (class A pbp2b gene) (4); line c, PRSP DN87/577 (class B pbp2b gene) (4); line d, PRSP 7026 (mosaic structure of the PBP 2B gene from S. mitis) (7); lines e through k, PRSP KK3, NA9, UB30, KY7, KK8, KS13, and OH01, respectively, as determined in the present study. Underlining indicates the sequence used as a probe for hybridization. Asterisks indicate the deletion of a nucleic acid.

FIG. 2. Deduced amino acid sequences of part of the PBPs 2B of the PSSP and PRSP strains listed in Fig. 1. The amino acid sequence of PSSP R6 is shown in line a. Only those sequences that differ from the R6 sequence are shown. Line b, PRSP 53139/72 possessing the class A pbp2b gene; line c, PRSP DN87/577 possessing the class B pbp2b gene; line d, PRSP 7026 possessing a mosaic structure pbp2b gene; lines e through g, PRSP KK3, NA9, and UB30, respectively, possessing the direct repeat sequences of the 9 nucleotides; lines h through j, PRSP KY7, KK8, and KS13, respectively, possessing substitutions near the conserved amino acid motifs; line k, PRSP OH01. The boxes in line a represent the conserved amino acid motifs. Underlining indicates the position used as a probe. Asterisks indicate the existence of a silent nucleotide mutation. The dot represents an amino acid deletion.

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TABLE 1. Serotypes, MICs of b-lactam antibiotics, and PBP affinities for penicillin of S. pneumoniae strains possessing the 9 direct repeat nucleotides in the pbp2b gene Strain

KK3 UB30 ME9 ME24 H18 NA9 KU17 KU27 KW13 HO12 ME34 H5 KS4

Serotype

19 19 19 19 19 19 19 19 6 19 19 6 6

TABLE 2. MICs of penicillin, dot blot hybridization results, and PBP affinities for penicillin of PRSP strains classified as unknown

Affinity of PBPsb

MIC (mg/ml)a

1259

Pc

Ctx

Czx

Papm

1A

2B

2.0 2.0 2.0 2.0 1.0 1.0 1.0 1.0 1.0 0.5 0.5 0.25 0.125

0.5 1.0 1.0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.125 0.063

16 16 16 16 16 8 16 16 8 4 8 0.25 0.125

0.125 0.125 0.125 0.063 0.125 0.063 0.125 0.063 0.125 0.063 0.125 0.016 0.063

2 2 2 2 2 2 2 2 2 2 2 1 1

2 2 2 2 2 2 2 2 2 2 2 2 2

a Abbreviations: Pc, benzylpenicillin; Ctx, cefotaxime; Czx, ceftizoxime; Papm, panipenem. b The affinities of PBPs for benzylpenicillin were assayed by previously described methods (13). 2, altered PBP affinity; 1, normal PBP affinity.

mg/ml for cefotaxime (Table 1). These strains were also confirmed to have the altered PBP 2B that reduces the affinity for penicillin, but the affinities of PBPs 1a, 2A, and 3 were normal. To evaluate the importance of the three substitutions common to five or six strains (Fig. 2), we conducted dot blot hybridization with 37 ‘‘unknown’’ strains. Three probes fully complementary to the PSSP R6 sequence were designed and labeled with biotin at the 59 end: (i) probe PSSP01 (59-TCAT CAAATACCTATATG; nucleotides 1,556 to 1,573), (ii) probe PSSP02 (59-GGAGAAACTGCGTTCAA; nucleotides 1,654 to 1,670), and (iii) probe PSSP03 (59-CTTGGGTACTGCGAC A; nucleotides 1,687 to 1,702). We then conducted conventional hybridization using a 382-bp DNA fragment amplified by primers P2B6 and P2B4 (59-AGTAGATTCATCTGGTAG GTC; nucleotides 1,709 to 1,729) and the three probes (9). In the control experiment with the sequenced strains, all probes gave positive signals for the OH01 strain, which has no mutations, but negative signals for the six mutated strains, which possess mismatches of two or three nucleotides. In the results of dot blot hybridization experiments, the MICs of penicillin, and the affinities of PBP 2B and PBP 1A for penicillin (Table 2), strains with no positive signals with all three probes, indicating the presence of some mutation(s) in these regions, were named type I. These strains had relatively higher levels of resistance to penicillin than strains of the other types did. The type II strain had a mutation(s) at two regions of PSSP02 and PSSP03, and the type III strain had a mutation(s) only at region PSSP01. The type IV strain showed no mutation in the three regions and was not confirmed to have an alteration in the affinity of its PBP 2B for penicillin. This suggests that substitutions at the three mutational sites near the conserved amino acid motifs were essentially associated with penicillin resistance, which is consistent with the results reported by Smith and Klugman (10). Previous studies with the Neisseria gonorrhoeae PBP 2 gene (3) have shown that the insertion of an extra amino acid between the two conserved motifs reduces the affinity of PBP 2 for penicillin. The insertion of three amino acids in this region that we have described here may have been responsible for the altered PBP 2B and may have been involved as one of the determinants of penicillin resistance.

Type

No. of strains

Benzylpenicillin MIC (mg/ml)

I II III IV

27 1 1 8

0.125–1 0.125 1 0.125–0.25

Dot blot hybridizationa

Affinity of PBPsb

PSSP01

PSSP02

PSSP03

1A

2B

2 1 2 1

2 2 1 1

2 2 1 1

2 or 1 NDc 2 2

2 ND 2 1

Dot blot hybridization result: 2, negative; 1, positive. The affinities of PBPs for benzylpenicillin were assayed by previously described methods (13). 2, altered PBP affinity; 1, normal PBP affinity. c ND, not determined. a b

Nucleotide sequence accession numbers. The partial sequences of the pbp2b genes from strains KK3 and NA9 in Fig. 1 have been submitted to the GSDB/DDBJ/EMBL/NCB database and have been assigned accession numbers D42074 and D43075, respectively. We thank the members of the Working Group for PRSP for collecting the strains used in the study. REFERENCES 1. Appelbaum, P. C. 1992. Antimicrobial resistance in Streptococcus pneumoniae: an overview. Clin. Infect. Dis. 15:77–83. 2. Barcus, V. A., K. Ghankar, M. Yeo, T. J. Coffey, and C. G. Dowson. 1995. Genetics of high level penicillin resistance in clinical isolates of Streptococcus pneumoniae. FEMS Microbiol. Lett. 126:299–303. 3. Brannigan, J. A., I. A. Tirodimos, Q. Y. Zhang, C. G. Dowson, and B. G. Spratt. 1990. Insertion of an extra amino acid is the main cause of the low affinity of penicillin-binding protein 2 in penicillin-resistant strains of Neisseria gonorrhoeae. Mol. Microbiol. 4:913–919. 4. Dowson, C. G., T. J. Coffey, C. Kell, and R. A. Whiley. 1993. Evolution of penicillin resistance in Streptococcus pneumoniae; the role of Streptococcus mitis in the formation of a low affinity PBP2B in S. pneumoniae. Mol. Microbiol. 9:635–643. 5. Dowson, C. G., A. Hutchison, J. A. Brannigan, R. C. George, D. Hansman, J. Lin ˜ ares, A. Tomasz, J. M. Smith, and B. G. Spratt. 1989. Horizontal transfer of penicillin-binding protein genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae. Proc. Natl. Acad. Sci. USA 86:8842– 8846. 6. Dowson, C. G., A. Hutchison, and B. G. Spratt. 1989. Extensive remodelling of the transpeptidase domain of penicillin-binding protein 2B of a penicillinresistant South African isolate of Streptococcus pneumoniae. Mol. Microbiol. 3:95–102. 7. Dowson, C. G., A. Hutchison, and B. G. Spratt. 1989. Nucleotide sequence of the penicillin-binding protein 2B gene of Streptococcus pneumoniae strain R6. Nucleic Acids Res. 17:7518. 8. Hultman, T., S. Stahl, E. Hornes, and M. Uhlen. 1989. Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support. Nucleic Acids Res. 17:4937–4946. 9. Saiki, R. K., C.-A. Chang, C. H. Levenson, T. C. Warren, C. D. Boehm, H. H. Kazazian, Jr., and H. A. Erlich. 1988. Diagnosis of sickle cell anemia and b-thalassemia with enzymatically amplified DNA and nonradioactive allelespecific oligonucleotide probes. N. Engl. J. Med. 319:537–541. 10. Smith, A. M., and K. P. Klugman. 1995. Alterations in penicillin-binding protein 2B from penicillin-resistant wild-type strains of Streptococcus pneumoniae. Antimicrob. Agents Chemother. 39:859–867. 11. Ubukata, K., S. Nakagami, A. Nitta, A. Yamane, S. Kawakami, M. Sugiura, and M. Konno. 1992. Rapid detection of the mecA gene in methicillinresistant staphylococci by enzymatic detection of polymerase chain reaction products. J. Clin. Microbiol. 30:1728–1733. 12. Ubukata, K., H. Nakano, A. Yamane, and M. Konno. 1995. Relationship of the tandem repeat sequences of the PBP-2B gene with the level of penicillin resistance in Streptococcus pneumoniae, abstr. C123, p. 62. In Program and abstracts of the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. 13. Ubukata, K., N. Yamashita, and M. Konno. 1985. Occurrence of a b-lactaminducible penicillin-binding protein in methicillin-resistant staphylococci. Antimicrob. Agents Chemother. 27:851–857.