Article Figures & Data
Figures
- FIG. 1.
Phylogeny of the K. oxytoca β-lactamase gene. The neighbor-joining tree was rooted using as an outgroup the gene sequence of the SFO-1 β-lactamase (GenBank accession no. AB003148 ), the closest relative of blaOXY. Values at the nodes correspond to bootstrap values obtained after 1,000 replicates. Six major branches of K. oxytoca β-lactamase sequences are visible, corresponding to the blaOXY-1 to blaOXY-6β -lactamase gene groups.
- FIG. 2.
Phylogeny obtained based on 1,896 nt positions from the three housekeeping genes rpoB, gyrA, and gapDH. The neighbor-joining tree was rooted using K. pneumoniae type strain SB132 (= ATCC 13883T). Values at the nodes correspond to bootstrap values obtained after 1,000 replicates. Five branches of K. oxytoca are distinguished, corresponding to phylogenetic groups KoI to KoVI. blaOXY-5-harboring strains fall into the KoI branch.
Tables
- TABLE 1.
K. oxytoca strains studied, β-lactamase (bla) gene sequences, housekeeping gene sequences, and their characteristicsa
No. Strain name(s) Geographical source Clinical source Phylogenetic group bla group bla accession no. Housekeeping gene accession no. rpoB gyrA gapDH 16S rRNA Study isolates 1 SB9 Spain Blood KoI (f) bla OXY-1 AJ871864 AJ871801 AJ303591 AJ871820 AJ871855 2 SB71 France Blood KoI (f) bla OXY-1 AJ871865 AJ871802 AJ871839 AJ871821 AJ871856 3 SB136 (ATCC 49131) KoII (f) bla OXY-2 AJ871866 AJ871803 AF303608 AJ871822 AJ871857 4 SB175 (ATCC 13182T) KoII (f) bla OXY-2 AJ871867 AJ871804 AJ871840 AJ871823 AJ871858 5 SG271 Clinical KoIII bla OXY-3 (a) AF491278 (a) AJ871805 AJ871842 AJ871824 NDb 6 SG266 Clinical KoIV bla OXY-4 (a) AY077481 (a) AJ871806 AJ871841 AJ871825 ND 7 SB2908 Italy Blood KoI bla OXY-5 AJ871868 AJ871807 AJ871843 AJ871826 AJ871859 8 SB2909 Italy Blood KoI bla OXY-5 AJ871869 AJ871808 AJ871844 AJ871827 ND 9 SB2910 Italy Blood KoI bla OXY-5 AJ871870 AJ871809 AJ871845 AJ871828 ND 10 SB2933 Switzerland Blood KoI bla OXY-5 AJ871871 AJ871810 AJ871846 AJ871829 ND 11 SB2942 Netherland Blood KoI bla OXY-5 AJ871872 AJ871811 AJ871847 AJ871830 AJ871860 12 SB73 France Wound KoVI bla OXY-6 AJ871873 AJ871812 AJ871848 AJ871831 AJ871861 13 SB75 Germany Blood KoVI bla OXY-6 AJ871874 AJ871813 AJ871849 AJ871832 ND 14 SB324 Germany Blood KoVI bla OXY-6 AJ871875 AJ871814 AJ871850 AJ871833 ND 15 SB352 France Wound KoVI bla OXY-8 AJ871876 AJ871815 AJ871851 AJ871834 ND 16 SB397 South Africa Blood KoVI bla OXY-6 AJ871877 AJ871816 AJ871852 AJ871835 ND 17 SB3037 Ukraine Wound KoVI bla OXY-6 AJ871878 AJ871817 AJ871853 AJ871836 AJ871862 18 SB3051 France Blood KoVI bla OXY-6 AJ871879 AJ871818 AJ871854 AJ871837 AJ871863 K. oxytoca c 19 SL781 (CIP104963) France KoI bla OXY-1 (b) Z30177 (b) ND ND ND ND 20 SG12 France KoI bla OXY-1 (a) AYO77482 (a) ND ND ND ND 21 SG337 France KoI bla OXY-1 (a) AYO77483 (a) ND ND ND ND 22 SG49 France KoI bla OXY-1 (a) AYO77486 (a) ND ND ND ND 23 KH66 Sweden KoI bla OXY-1 (c) Y17715 (c) ND ND ND ND 24 E23004 Japan KoI bla OXY-1 (d) M27459 (d) ND ND ND ND 25 SG77 France KoII bla OXY-2 (a) AY077488 (a) ND ND ND ND 26 SG43 France KoII bla OXY-2 (a) AY077487 (a) ND ND ND ND 27 SG176 France KoII bla OXY-2 (a) AY077485 (a) ND ND ND ND 28 (CIP106098) SL911 France KoII bla OXY-2 (b) Z49084 (b) ND ND ND ND 29 SC10,436 KoII bla OXY-2 (e) AY055205 (e) ND ND ND ND 30 KH11 Sweden KoII bla OXY-2 (c) Y17714 (c) ND ND ND ND K. pneumoniae 31 SB132 (ATCC 13883T) KpI bla SHV (g) AJ635407 (g) AJ871819 AF303606 (f) AJ875018 ND ↵ a Phylogenetic groups, bla groups, and bla accession numbers followed by letter in parentheses were previously determined. References: a, 14; b, 11; c,31; d,1; e,15a; f,4; g,18. Other accession numbers correspond to sequences determined in this work.
↵ b ND, not determined.
↵ c Nucleotide sequences included for comparison.
- TABLE 2.
Primers, their sequences, and their positions on the gene
Gene and primer Sequence Positionsa bla OXY-E 5′-GGT TTT GGT AAC TGT GAC GGG-3′ −189 to −168 OXY-G 5′-CAG AGT GCA GAG TGT TGC AG-3′ 913 to 894 OXY-H 5′-GCG ACA ATA C(G/C)G CGA TGA-3′ 397 to 414 OXY1-A 5′-CGT GGC GTA AAA CCG CCC TG-3′ 14 to 33 OXY1-B 5′-GTC CGC CAA GGT AGC TAA TC-3′ 442 to 423 OXY2-A 5′-AAG GCT GGA GAT TAA CGC AG-3′ 285 to 304 OXY2-B 5′-GCC CGC CAA GGT AGC CGA TG-3′ 439 to 420 OXY5-B 5′-GAT TAA AAA AGC GGA TTT AGT A-3′ 297 to 318 OXY5-C 5′-GCC CGC CAA GGT AC CGA TA-3′ 442 to 423 OXY6-B 5′-CGG ATG ATT CGC AAA CCC TT-3′ 167 to 186 OXY6-C 5′-CGG GAT GGC TTT CGC TCT GC-3′ 274 to 255 rpoB VIC2 5′-GGT TAC AAC TTC GAA GAC TC-3′ 2469 to 2489 VIC3 5′-GGC GAA ATG GC(A/T) GAG AAC CA-3′ 1442 to 1422 gyrA gyrA-A 5′-CGC GTA CTA TAC GCC ATG AAC GTA-3′ 139 to 163 gyrA-C 5′-ACC GTT GAT CAC TTC GGT CAG G-3′ 762 to 741 gapDH gapF-173 5′-TGA AAT ATG ACT CCA CTC ACG G-3′ 135 to 153 gapR-181 5′-CTT CAG AAG CGG CTT TGA TGG CTT-3′ 771 to 747 rrs Ad 5′-AGA GTT TGA TCM TGG CTC AG-3′ 8 to 27 rJ 5′-GGT TAC CTT GTT ACG ACT T-3′ 1508 to 1491 D 5′-CAG CAG CCG CGG TAA TAC-3′ 518 to 535 E 5′-ATT AGA TAC CCT GGT AGT CC-3′ 786 to 805 rE 5′-GGA CTA CCA GGG TAT CTA AT-3′ 805 to 786 - TABLE 3.
Amino acid sequence variation among OXY-1 to OXY-6 β-lactamase (bla) groups
Strain bla allelea Enzymea pI Amino acid at position (Ambler no.): Amino acid at position (Ambler no.): Amino acid at position (Ambler no.): Amino acid at position (Ambler no.): 2 4 9 (5) 11 (7) 12 (8) 15 (11) 16 (12) 17 (13) 22 (18) 24 (20) 25 (21) 29 (25) 30 (26) 31 (27) 32 (28) 33 (29) 34 (30) 38 (34) 39 (35) 43 (39) 44 (40) 54 (50) 56 (52) 58 (54) 61 (56) 75 (72) 90 (87) 91 (88) 92 (89) 101 (98) 102 (99) 103 (100) 111 (108) 129 (126) 140 (137) 141 (138) 143 (140) 156 (153) 161 (158) 168 (165) 171 (168) 174 (171) 180 (177) 184 (181) 194 (190) 200 (197) 230 (227) 233 (230) 240 (237) 256 (255) 261 (260) 262 (261) 270 (269) 275 (274) 278 (277) SL781 bla OXY-1-1a OXY-1-1 7.5b L S T L M A A V A G S S A D A I Q A D R S N A D T G N P E K K S I A K M S Q V T A S K T R N A A A N V L Q S E SB71 bla OXY-1-2a OXY-1-2 7.7 V G SB9 bla OXY-1-2a OXY-1-2 7.7 V G SG12 bla OXY-1-3a OXY-1-3 NAc G SG337 bla OXY-1-4a OXY-1-4 NA Deld V SG49 bla OXY-1-5a OXY-1-5 NA V G KH66 bla OXY-1-6a OXY-1-6 5.25b P K E23004 bla OXY-1-7a OXY-1-7 7.4b SG271 bla OXY-3-1a OXY-3-1 6.7b I T S I Del L C S Del Del N S T D T N I K D A V G R T E H V L D SG266 bla OXY-4-1a OXY-4-1 7.7b S Del T L H D I G M G SB2908 bla OXY-5-1a OXY-5-1 7.2 Del H T A I G M SB2909 bla OXY-5-1a OXY-5-1 7.2 Del H A I G M SB2910 bla OXY-5-1a OXY-5-1 7.2 Del H A I G M SB2942 bla OXY-5-1a OXY-5-1 7.2 Del H A I G M SB2933 bla OXY-5-2a OXY-5-2 7.7 Del A I G L M SB73 bla OXY-6-1a OXY-6-1 7.75 S H D SB75 bla OXY-8-1a OXY-6-1 7.75 S H D SB324 bla OXY-6-2a OXY-6-2 7.75 S Del H D SB352 bla OXY-5-3a OXY-6-3 7.75 S T H D SB397 bla OXY-6-4a OXY-6-4 8.1 S N H D SB3037 bla OXY-6-4a OXY-6-4 8.1 S N H D SB3051 bla OXY-6-4a OXY-6-4 8.1 S N H D SB136 bla OXY-2-1a OXY-2-1 NA I I M L Del A T H T N I S K N A A T L I G H A T T E S D E V D N SB175 bla OXY-2-2a OXY-2-2 NA I I M L Del A T H T N I S K N A A T L I G R A T T E S D E V D I N SG77 bla OXY-2-3a OXY-2-3 NA I I M L Del Del A T H T N I S K N A A T L I G R A T T E S D E V G D I N KH11 bla OXY-2-4a OXY-2-4 NA I I M L Del Del A T H T N I S K N A A T L I G R A T T E S D E V D N SG43 bla OXY-2-5a OXY-2-5 NA I I M L Del Del A T H T N I S K N A A T L I G R A T T E S D E V D N SG176 bla OXY-2-6a OXY-2-6 NA I I M L Del S A T H T N I S K N A A T L I G R A T T E S D E V D N SL911 bla OXY-2-7a OXY-2-7 NA I I M L Del A T H T N I S K N A A T L I G R A T T E S D E V D N SC10,436 bla OXY-2-8a OXY-2-8 NA I I M L Del S A T H T N I S K N A A T L I G R A T T E S A E V N - TABLE 4.
Percent average nucleotide and aminoacid β-lactamase sequence similarity within and among bla OXY groupsa
Group (no. of strains) Avg % similarity (SD) blaOXY-1 blaOXY-2 blaOXY-3 blaOXY-4 blaOXY-5 blaOXY-6 bla OXY-1 (8) 99.7 (0.09)/99.4 (0.4) 87.2 (2.3) 86.2 (5) 95.2 (1.7) 97.5 (0.5) 97.1 (0.5) bla OXY-2 (8) 89.5 (0.4) 99.7 (0.1)/99.4 (0.4) 84.9 (5.6) 86.2 (5) 87 (2.9) 86.5 (2.6) bla OXY-3 (1) 90.9 (0.2) 88.6 (0.5) 100 (0)/100 (0) 86.3 (0.0) 85.1 (6.7) 85.6 (5.6) bla OXY-4 (1) 96.8 (0.3) 88.3 (0.3) 90 (0) 100 (0)/100 (0) 95.9 (1.7) 95.6 (0.1) bla OXY-5 (5) 97.5 (0.3) 91.5 (0.3) 90.3 (0) 97.2 (0.4) 99.8 (0.1)/99.7 (0.4) 95.8 (1) bla OXY-6 (7) 98.3 (0.3) 88.7 (0.3) 91.2 (0.3) 97.7 (0.2) 97 (0.2) 99.8 (0.1)/99.7 (0.2) ↵ a Above diagonal, nucleotide sequence similarity; below diagonal, amino acid sequence similarity; inside diagonal cells: nucleotide/amino acid sequence similarities.
- TABLE 5.
MICs of 10 β-lactams and threeβ -lactam-β-lactam inhibitor combinations observed for strains harboring blaOXY-1 to blaOXY-6 β-lactamase genes
Drug(s) MIC (μg/ml) blaOXY-1 strains SB9, SB71 blaOXY-2 strains SB136, SB175 blaOXY-3 strains SG271a blaOXY-4 strains SG266a blaOXY-5 strains SB2908, SB2933 blaOXY-6 strains SB73, SB324, SB352, SB397 Amoxicillin 256 512 16 64 256 256 Amoxicillin + 2 μg/ml of clavulanate 4 4 0.25 0.5 4 2-4 Ticarcillin 128 128 16 64 128 64-128 Ticarcillin + 2 μg/ml of clavulanate 2 2 0.5 2 2 1-2 Piperacillin 16-32 16-32 1 2 8-32 8-32 Piperacillin + 4 μg/ml of tazobactam 2 1-2 0.12 0.5 2-4 2-4 Cephalothin 4-8 16 2 2 2-4 4-8 Cefoxitin 4-8 2 0.5 1 2-8 4-8 Cefotaxime ≤0.06 ≤0.06-0.125 ≤0.06 ≤0.06 ≤0.06 ≤0.06 Ceftazidime 0.125-0.25 0.125-0.25 ≤0.06 ≤0.06 0.125-0.25 0.25-0.5 Cefepime ≤0.06 ≤0.06-0.125 ≤0.06 ≤0.06 ≤0.06 ≤0.06 Aztreonam ≤0.06-0.25 0.25 ≤0.06 ≤0.06 ≤0.06-0.125 ≤0.06-0.25 Imipenem 0.125 0.125 0.5 0.5 0.125 0.125 ↵ a Values previously published (14).
- TABLE 6.
Nucleotide polymorphism observed among the 18 study isolates
Gene Fragment size (bp) No. of variable sites (%) No. of substitutions (%) Singleton Informative Total Synonymous Nonsynonymous Total rpoB 940 34 (3.6) 48 (5.1) 82 (8.7) 82 (8.7) 5 (0.5) 87 (9.3) gyrA 383 15 (3.9) 17 (4.4) 32 (8.4) 33 (8.6) 1 (0.3) 34 (8.9) gapDH 573 26 (4.5) 13 (2.3) 39 (6.8) 38 (6.6) 3 (0.5) 41 (7.2) bla 870, 873, or 876 64 (7.3) 139 (15.9) 203 (23.2) 158 (18.0) 52 (5.9) 210 (24.0) - TABLE 7.
Percent average rpoB, gyrA, and gapDH nucleotide sequence divergence within and among the five K. oxytoca phylogenetic groups and clone blaOXY-5
Group or clone Avg % divergence from nucleotide sequence KoI KoII KoIII KoIV blaOXY-5 clone KoVI rpoB gyrA gapDH rpoB gyrA gapDH rpoB gyrA gapDH rpoB gyrA gapDH rpoB gyrA gapDH rpoB gyrA gapDH KoI 0.2 0.5 0 3.09 3.2 1.92 4.25 4.96 4.36 3.2 3.65 0.7 0.49 0.52 0.00 3.04 2.68 0.67 KoII 0.11 0.13 0 4.15 5.09 5.4 4.15 3.26 2.44 3 3.13 1.92 3.6 2.68 2.24 KoIII NAa NA NA 4.36 5.48 4.88 4.32 4.7 4.36 4.32 4.51 4.51 KoIV NA NA NA 2.83 3.65 0.7 2.96 1.3 0.67 bla OXY-5 clone 0.17 0.00 0.06 3.1 2.42 0.67 KoVI 0.4 0.13 0.1 ↵ a NA, not applicable (only one sequence per group).
- TABLE 8.
Average numbers of synonymous substitutions per synonymous site (Ks values) among the five K. oxytoca phylogenetic groups and clone blaOXY-5a
Group or clone Avg Ks value KoI KoII KoIII KoIV Clone blaOXY-5 KoVI KoI 0.114 0.17 0.128 0.02 0.12 KoII 0.135 0.157 0.156 0.111 0.133 KoIII 0.21 0.214 0.175 0.173 0.171 KoIV 0.143 0.126 0.22 0.113 0.117 Clone blaOXY-5 0.022 0.132 0.199 0.143 0.123 KoVI 0.112 0.128 0.188 0.045 0.101 ↵ a Data computed from gene rpoB (above diagonal) and gyrA (below diagonal).
