Commensal Streptococci Serve as a Reservoir for β-Lactam Resistance Genes in Streptococcus pneumoniae

Anders Jensen; Oskar Valdórsson; Niels Frimodt-Møller; Susan Hollingshead; Mogens Kilian

doi:10.1128/AAC.00429-15

DOI: 10.1128/AAC.00429-15

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FIG 1
(a to c) Comparative analyses of pbp genes in Mitis group streptococci. Left columns show minimum evolution trees generated in MEGA of the transpeptidase region of pbp2x (a), pbp2b (b), and pbp1a (c) genes of susceptible and nonsusceptible strains of commensal streptococci and S. pneumoniae. Bootstrap values of 500 replications are shown. Clusters CX1 to CX8 (pbp2x), CB1 to CB5 (pbp2B), and CA1 to CA9 (pbp1a) identified by the BratNextgen software for detecting recombination are highlighted. In the pbp2x tree, +, −, or (−) after a strain name denotes the presence of, absence of, or a nonfunctional murM gene. The middle columns show recombination segments identified by the BratNextgen software over the length of the sequenced part of the transpeptidase region of the pbp genes. Blocks with the same color are hypothesized to be of the same origin. The numbers at the bottom of the middle columns indicate the region of the genes used for the analysis according to the positions in sequences of S. pneumoniae R6. Right columns show the susceptibility of six beta-lactam antibiotics (benzylpenicillin [pen], piperacillin [pip], oxacillin [oxa], cephalothin [cep], cefuroxime [cef], and cefotaxime [ctx]) indicated as susceptible (green), intermediate (yellow), and resistant (red) according the thresholds given by EUCAST (28) and shown in Table 2.
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FIG 2
(a to c) Codons with amino acid changes in the transpeptidase region of PBP2x (a), PBP2b (b), and PBP1a (c) compared to the S. pneumoniae R6 reference strain. Only amino acids that differ from the reference sequence of the R6 strain are shown. Percentages of isolates in each group that have amino acid changes in the specified position are indicated. Amino acid positions further marked with colors or an asterisk indicate sites previously associated with resistance in S. pneumoniae and commensal streptococci (see reference 8). Orange, laboratory mutants; green, clinical isolates; purple, both laboratory mutants and clinical isolates. Amino acid alterations linked to reduced beta-lactam susceptibility in a study by Chewapreecha et al. (21) are marked with an asterisk. The color scale bar represents the percentage of isolates with altered amino acids compared to S. pneumoniae R6 in each position.

TABLE 1

List of primers used in this study and sequence information

Gene(s)	Primer name	Primer sequence (5′ to 3′)	Position(s)^a	Trimmed amino acid sequence (positions)^b	No. (%) of variable nucleotide sites/total no. of nucleotide sits^f	No. (%) of variable amino acid sites/total no. of amino acid sites^f
pbp1a	pbp1a-f	ACIACDGGDATGGAHGTHTA	877–896	318–577	371/780 (48)	98/263(37%)
pbp1a	pbp1a-r	GTCCADACRGCCATWGMATA	1790–1771
pbp2b	pbp2b-f	AATGAYCGHGTBGGDACYTC	793–812	440–643	294/612 (48)	79/204 (39)
pbp2b	pbp2b-r	TTGATRATRTCRCGHGCAAT	2027–2008
pbp2x	pbp2x-f	AAGTAYATGACIGCDACCTT	862–881	308–554	320/741 (43)	79/247 (32)
pbp2x	pbp2x-r	TGVAGRTTRAGRGADTCTTT	1865–1846
murM and murN	murMN-f	CAYGARTGGTAYTAYTGGGAA	694–714^c
murM and murN	murMN-r	TGAATRTARCAWCCAGGRCT	62–43^d
Ortholog of murM	murM_uo5-f	TTGCARAGTAGTGATTGGKCCA	76–97^e
Ortholog of murM	murM_uo5-r	TCCTTTATTYYTTGCAGTTCGGA	556–534^e

↵a Nucleotide positions according to the S. pneumoniae R6 sequence.
↵b Amino acid positions in S. pneumoniae R6. Active sites range from residue 337 to residue 549 (pbp2x), 386 to 617 (pbp2b), and 370 to 557 (pbp1a).
↵c Nucleotide position in the flanking SP_0614 gene in S. pneumoniae R6 of the mugsy cluster of murM and murN.
↵d Nucleotide position in the flanking SP_0618 gene in S. pneumoniae R6 of the mugsy cluster of murM and murN.
↵e Nucleotide position in the ortholog of the murM gene in S. oralis uo5.
↵f Numbers of all variable sites for all strains included in the sequence analysis.

TABLE 2

MICs of 63 strains of commensal streptococci divided by species designation

Antibiotic and MIC (μg/ml)	No. (%) of strains	No. (%) of strains of species:
Antibiotic and MIC (μg/ml)	No. (%) of strains	S. mitis (n = 46)	S. oralis (n = 10)	S. infantis (n = 6)	S. pseudopneumoniae (n = 1)
Benzylpenicillin
≤0.25	30 (48)	21 (46)	7 (70)	1 (17)	1
>0.25–2	11 (17)	9 (19)	1 (10)	1 (17)
>2	22 (35)	16 (35)	2 (20)	4 (66)
Median	1.0	1.0	0.1875	4.0
Piperacillin^a
≤0.25	22 (35)	15 (33)	6 (60)		1
>0.25–2	15 (24)	11 (24)	1 (10)	3 (50)
>2	26 (41)	20 (43)	3 (30)	3 (50)
Mean	1.0	1.0	0.125	2.5
Oxacillin^a
≤0.25	15 (24)	12 (26)	2 (20)		1
>0.25–2	15 (24)	10 (22)	4 (40)	1 (16)
>2	33 (52)	24 (52)	4 (40)	5 (84)
Median	4.0	4.0	1.25	12.0
Cephalothin^b
≤0.5	16 (25)	14 (30)	1 (10)		1
>0.5–2	10 (16)	5 (11)	4 (40)	1 (16)
>2	37 (59)	27 (59)	5 (50)	5 (84)
Median	8.0	4.0	3.0	8.0
Cefuroxime
≤0.5	24 (39)	18 (40)	5 (50)		1
>0.5–1	1 (2)			1 (16)
>1	37 (59)	27 (60)	5 (50)	5 (84)
Median	4.0	4.0	1.25	4.0
Cefotaxime
≤0.5	27 (44)	20 (44)	6 (60)		1
>0.5–2	18 (29)	11 (25)	2 (20)	5 (84)
>2	17 (27)	14 (31)	2 (20)	1 (16)
Median	2.0	2.0	0.282	2.0

↵a No resistant breakpoints for the mitis group of streptococci are given by EUCAST. Breakpoints for benzylpenicillin are used for comparison.
↵b No resistant breakpoints for the mitis group of streptococci are given by EUCAST. Breakpoints for cefotaxime are used for comparison.

Additional Files

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Tables

Supplemental material

Supplemental file 1 -
Supplemental Table S1: strain information.

XLSX, 42K

Supplemental file 2 -
Supplemental Table S2: amino acid comparison for PBP2x.

XLSX, 57K

Supplemental file 3 -
Supplemental Table S3: amino acid comparison for PBP2b.

XLSX, 48K

Supplemental file 4 -
Supplemental Table S4: amino acid comparison for PBP1a.

XLSX, 51K