Introduction
Methodology
Bacterial growth and culture conditions
Minimum inhibitory concentration (MIC)
In vitro clarithromycin resistance induction
Random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR)
Sanger sequencing
Library preparation, and sequencing
Single-nucleotide variations (SNVs) and insertion and deletions (InDels) identification
Probability of mutation occurrence
Growth curve
Biofilm formation
Statistical analysis
Results
Clarithromycin-resistant isogenic isolates
No | Isolate | CLR sensitivity/resistance | CLR MIC (µg/mL) | 23S rRNA (A2142G/C) | 23S rRNA (A2143G) | Induction success |
---|---|---|---|---|---|---|
1 | NCTC 11637 | S | 0.032 | No | No | No |
2 | UM025B | S | 0.032 | No | No | No |
3 | UM067B SW | S | 0.125 | No | No | No |
4 | UM113B | S | 0.125 | No | No | No |
5 | UM113B C5 | S | 0.032 | No | No | No |
6 | UM114 | S | 0.032 | No | No | No |
7 | UM171 | S | 0.064 | No | Yes | Yes |
8 | UM276 R (71) | S | 0.032 | No | No | No |
9 | UM276 S (53) | S | 0.032 | No | No | No |
10 | UM303A C6 | S | 0.032 | No | No | No |
11 | UM408 SW | S | 0.032 | No | No | No |
12 | UM411 SW | S | 0.032 | No | No | No |
13 | UM443A (33) | S | 0.032 | No | No | No |
14 | UM443A SW | S | 0.032 | No | No | No |
15 | UM622A1a | S | 0.032 | No | No | No |
16 | UM626A1 | S | 0.064 | No | Yes | Yes |
17 | UM650B | S | < 0.016 | No | Yes | Yes |
18 | UM663B2 | S | 0.064 | No | No | No |
19 | UM678A | S | 0.064 | No | Yes | Yes |
20 | UM692B2 | S | 0.032 | No | No | No |
21 | UM113A SW | S | 0.032 | No | No | No |
22 | H. pylori 26695 | S | 0.016 | No | No | Not Done |
23 | UM032 | S | 0.016 | No | No | Not Done |
24 | UM202 | R | – | No | No | Not Done |
25 | UM233 | S | – | No | No | Not Done |
26 | UM370 | R | – | No | Yes | Not Done |
No | Isolate | MIC in µg/mL (passage number) | |||
---|---|---|---|---|---|
S isolates before induction | B isolates at breakpoint | R isolates after breakpointa | R isolates after resistance inductionb | ||
1 | UM171 | 0.064 (P0) | 0.125 (P4) | 1 (P5) | > 64 (P10) |
2 | UM626A1 | 0.064 (P0) | 0.125 (P5) | 2 (P6) | > 64 (P11) |
3 | UM650B | < 0.016 (P0) | 0.125 (P7) | 1 (P8) | > 64 (P12) |
4 | UM678A | 0.064 (P0) | 0.125 (P5) | 1 (P6) | > 64 (P10) |
Stability of resistance
RAPD genotypes
23SrRNA genotypes and resistance to clarithromycin
Quality of H. pylori genomes
Clarithromycin resistance-associated variants
SNV and InDel
Isolate | Group | Isolate ID | SNV | InDel | ||||||
---|---|---|---|---|---|---|---|---|---|---|
Non-synonymousa | Synonymousa | Stop gain | Stop loss | Non-frameshift deletion | Frameshift deletion | Non-frameshift insertion | Frameshift insertion | |||
UM171 | B | UM171-B | 7006 | 15,900 | 71 | 18 | 133 | 420 | 115 | 409 |
R | UM171-R | 6764 | 15,358 | 70 | 19 | 136 | 411 | 112 | 395 | |
UM626A1 | B | UM626A1-B | 9101 | 21,425 | 83 | 27 | 169 | 510 | 135 | 485 |
R | UM626A1-R | 8635 | 20,422 | 78 | 27 | 148 | 465 | 139 | 443 | |
UM650B | B | UM650B-B | 1432 | 2978 | 20 | 3 | 52 | 136 | 38 | 139 |
R | UM650B-R | 1504 | 3108 | 18 | 3 | 53 | 136 | 40 | 143 |
Isolate | Group | Isolate ID | Categories | |||||
---|---|---|---|---|---|---|---|---|
Exonic | Intergenic | Upstream | Downstream | Upstream; downstream | UTR5a | |||
UM171 | B | UM171-B | 24,072 | 320 | 953 | 1536 | 1881 | 1 |
R | UM171-R | 23,265 | 327 | 938 | 1479 | 1898 | 0 | |
UM626A1 | B | UM626A1-B | 31,935 | 559 | 1352 | 2032 | 2541 | 2 |
R | UM626A1-R | 30,357 | 533 | 1375 | 1998 | 2454 | 2 | |
UM650B | B | UM650B-B | 4798 | 61 | 202 | 440 | 445 | 0 |
R | UM650B-R | 5005 | 88 | 175 | 342 | 483 | 1 |
Genes associated with virulence and antibiotic resistance
Locus tag | Protein ID | Protein name | Isolate | Rate of mutation | Threshold | Probability of mutation occurrence |
---|---|---|---|---|---|---|
K747_RS06340 | WP_015644956.1 | Sel1 repeat family protein | UM171-B | 0.1054 | 0.0180 | 5.8387 |
UM171-R | 0.0948 | 0.0175 | 5.4160 | |||
UM626A1-B | 0.0948 | 0.0241 | 3.9339 | |||
UM626A1-R | 0.0948 | 0.0230 | 4.1162 | |||
UM650B-B | 0.0141 | 0.0037 | 3.7658 | |||
UM650B-R | 0.0070 | 0.0038 | 1.8372 | |||
K747_RS06360 | WP_015644959.1 | Transglycosylase SLT domain-containing protein (Cag4) | UM171-B | 0.0180 | 0.0180 | 6.4219 |
UM171-R | 0.0175 | 0.0175 | 5.3848 | |||
UM626A1-B | 0.0241 | 0.0241 | 3.8298 | |||
UM626A1-R | 0.0230 | 0.0230 | 4.0073 | |||
UM650B-B | 0.0037 | 0.0037 | 16.3223 | |||
UM650B-R | 0.0038 | 0.0038 | 15.9259 | |||
K747_RS06805 | WP_015645027.1 | Vacuolating cytotoxin domain-containing protein | UM171-B | 0.0568 | 0.0180 | 3.1475 |
UM171-R | 0.0509 | 0.0175 | 2.9048 | |||
UM626A1-B | 0.0586 | 0.0241 | 2.4298 | |||
UM626A1-R | 0.0591 | 0.0230 | 2.5651 | |||
UM650B-B | 0.0182 | 0.0037 | 4.8890 | |||
UM650B-R | 0.0215 | 0.0038 | 5.6153 | |||
K747_RS07265 | WP_015645103.1 | 16S rRNA (cytosine(1402)-N(4))-methyltransferase (RsmH) | UM171-B | 0.0335 | 0.0180 | 1.8547 |
UM171-R | 0.0432 | 0.0175 | 2.4666 | |||
UM626A1-B | 0.0443 | 0.0241 | 1.8364 | |||
UM626A1-R | 0.0292 | 0.0230 | 1.2654 | |||
UM650B-B | 0.0076 | 0.0037 | 2.0259 | |||
UM650B-R | 0.0108 | 0.0038 | 2.8239 |
Type | Locus tag (gene)/protein | Position | Type of variation | Bases change (amino acid change) | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
UM171 | UM626A1 | UM650B | ||||||||||
S | B | R | S | B | R | S | B | R | ||||
SNV | K747_RS06360 (cag4)/transglycosylase SLT domain-containing protein | NC_021215.3:1292240 | Non-synonymous, exonic | G (R) | G>A (R47K) | G>A (R47K) | G (R) | G>A (R47K) | G>A (R47K) | G (R) | G>A (R47K) | G>A (R47K) |
NC_021215.3:1292491 | Non-synonymous, exonic | G (V) | G>A (V131M) | G>A (V131M) | G (V) | G>A (V131M) | G>A (V131M) | G (V) | G>A (V131M) | G>A (V131M) | ||
NC_021215.3:1292500 | Non-synonymous, exonic | A (I) | A>T (I134L) | A>T (I134L) | A (I) | A>T (I134L) | A>T (I134L) | A (I) | A>T (I134L) | A>T (I134L) | ||
K747_RS07265(rsmH)/16S rRNA (cytosine(1402)-N(4))-methyltransferase | NC_021215.3:1499504 | Non-synonymous, exonic | T (S) | T (S) | T>G (S244A) | T (S) | T>G (S244A) | T>G (S244A) | T (S) | T (S) | T>G (S244A) | |
NC_021215.3:1499056 | Non-synonymous, exonic | T (N) | T (N) | T>A (N94K) | T (N) | T>A (N94K) | T>A (N94K) | T (N) | T>A (N94K) | T>A (N94K) | ||
NC_021215.3:1499175 | Non-synonymous, exonic | A (D) | A (D) | A>G (D134G) | A (D) | A>G (D134G) | A>G (D134G) | A (D) | A>G (D134G) | A>G (D134G) | ||
NC_021215.3:1499345 | Non-synonymous, exonic | C (L) | C (L) | C>T (L191F) | C (L) | C>T (L191F) | C>T (L191F) | C (L) | C>T (L191F) | C>T (L191F) | ||
K747_RS06340/Sel1 repeat family protein | NC_021215.3:1289818 | Non-synonymous, exonic | C (D) | C>T (D39N) | C>T (D39N) | C (D) | C>T (D39N) | C>T (D39N) | C (D) | C>T (D39N) | C>T (D39N) | |
InDel | K747_RS06360 (cag4)/transglycosylase SLT domain-containing protein | NC_021215.3:1292229_1292232 | Frameshift deletion, exonic | AGTG (E) | – (E43fs) | AGTG (E) | AGTG (E) | – (E43fs) | – (E43fs) | AGTG (E) | – (E43fs) | – (E43fs) |
NC_021215.3:1292233_1292234 | Frameshift insertion, exonic | – (V) | ATAA (V45fs) | – (V) | – (V) | ATAA (V45fs) | ATAA (V45fs) | – (V) | ATAA (V45fs) | ATAA (V45fs) | ||
K747_RS06805/vacuolating cytotoxin domain-containing protein | NC_021215.3:1393215_1393219 | Frameshift deletion, exonic | ACTAG (L) | – (L2430fs) | ACTAG (L) | ACTAG (L) | – (L2430fs) | – (L2430fs) | ACTAG (L) | – (L2430fs) | – (L2430fs) | |
NC_021215.3:1393222_1393223 | Frameshift insertion, exonic | – (S) | CAAAC (S2433fs) | – (S) | – (S) | CAAAC (S2433fs) | CAAAC (S2433fs) | – (S) | CAAAC (S2433fs) | CAAAC (S2433fs) |
Growth curves
Biofilm assessment
Isolate | Group | Sample ID | Average OD595 nm reading | Fold difference | ||
---|---|---|---|---|---|---|
B vs S (p-value) | R vs S (p-value) | R vs B (p-value) | ||||
UM171 | S | UM171-S | 0.14 | 2.07 (< 0.05) | 2.36 (< 0.05) | 1.14 (≥ 0.05) |
B | UM171-B | 0.29 | ||||
R | UM171-R | 0.33 | ||||
UM626A1 | S | UM626A1-S | 0.22 | 1.39 (< 0.05) | 2.03 (< 0.05) | 1.45 (< 0.05) |
B | UM626A1-B | 0.36 | ||||
R | UM626A1-R | 0.49 | ||||
UM650B | S | UM650B-S | 0.21 | 1.86 (< 0.05) | 2.19 (< 0.05) | 1.18 (< 0.05) |
B | UM650B-B | 0.40 | ||||
R | UM650B-R | 0.47 | ||||
UM678A | S | UM678A-S | 0.19 | 1.95 (< 0.05) | 2.63 (< 0.05) | 1.35 (< 0.05) |
B | UM678A-B | 0.38 | ||||
R | UM678A-R | 0.52 |
Identification of genes associated with biofilm formation
Type | Locus tag (gene)/protein | Position | Type of variation | Bases change (amino acid change) | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
UM171 | UM626A1 | UM650B | ||||||||||
S | B | R | S | B | R | S | B | R | ||||
SNV | K747_RS04025(hypE)/hydrogenase expression/formation protein HypE | NC_021215.3:820809 | Non-synonymous, exonic | C (G) | C>G (G145A) | C>G (G145A) | C (G) | C>G (G145A) | C>G (G145A) | C (G) | C>G (G145A) | C>G (G145A) |
NC_021215.3:821112 | Non-synonymous, exonic | T (H) | T>C (H44R) | T>C (H44R) | T (H) | T>C (H44R) | T>C (H44R) | T (H) | T>C (H44R) | T>C (H44R) | ||
NC_021215.3:821113 | Non-synonymous, exonic | G (H) | G>T (H44N) | G>T (H44N) | G (H) | G>T (H44N) | G>T (H44N) | G (H) | G>T (H44N) | G>T (H44N) | ||
K747_RS04030(hypF)/carbamoyltransferase HypF | NC_021215.3:822687 | Non-synonymous, exonic | C (A) | C>T (A271T) | C>T (A271T) | C (A) | C T (A271T) | C>T (A271T) | C (A) | C>T (A271T) | C>T (A271T) | |
NC_021215.3:822837 | Non-synonymous, exonic | C (D) | C>T (D221N) | C>T (D221N) | C (D) | C>T (D221N) | C>T (D221N) | C (D) | C>T (D221N) | C>T (D221N) | ||
K747_RS06380/Cag pathogenicity island protein | NC_021215.3:1300640 | Non-synonymous, exonic | T (K) | T>G (K608Q) | T>G (K608Q) | T (K) | T>G (K608Q) | T>G (K608Q) | T (K) | T>G (K608Q) | T>G (K608Q) | |
NC_021215.3:1300687 | Non-synonymous, exonic | C (S) | C>T (S592N) | C>T (S592N) | C (S) | C>T (S592N) | C>T (S592N) | C (S) | C>T (S592N) | C>T (S592N) |