Detection of point mutations associated with clarithromycin resistance and determination of biopsies with susceptible and resistant genotypes

A small biopsy fragment was digested in 20 µl of proteinase K (Qiagen SA, Courtaboeuf, France) with 180 µl of lysis buffer (Qiagen). DNA extraction was performed by using a MagNA Pure LC DNA isolation kit I (Qiagen). Detection of H.pylori and mutations in the 23S rRNA gene associated with clarithromycin resistance was performed by real-time PCR, as previously described [14]. The method included amplification of a fragment of the H.pylori 23S rRNA gene coupled with a simultaneous detection of the amplicon by probe hybridization, followed by a melting curve analysis of the amplicons which allows the observation of resistant and susceptible genotypes [15][16]. This PCR also enabled the identification of biopsies containing both susceptible and resistant genotypes. Finally, strains from two patients with susceptible and resistant genotypes were obtained and named 3657-S/3657-R and 3695-S/3695-R.

Culture and separation of susceptible and resistant mutants

In each clinical case with both susceptible and resistant genotypes (3657-S/3657-R and 3695-S/3695-R), biopsies were cultured on Mueller Hinton agar plates supplemented with 10% sheep blood and Polyvitex in a microaerobic atmosphere at 37˚C. Clarithromycin resistant mutants were selected by plating 10⁹ colony forming units (CFU) on the same medium containing 1 µg/ml of clarithromycin and clarithromycin susceptible strains were selected by plating 10⁹ CFU on the medium without clarithromycin.In both cases, each colony was subcultured and the minimal inhibitory concentrations (MIC) of clarithromycin against H.pylori were determined by agar diffusion using E-test clarithromycin strips and real-time PCR in order to confirm susceptibility or resistance. Resistant mutants and susceptible isolates were single-colonies purified on a selective plate and then immediately frozen at −80˚C.

Random amplified polymorphism DNA(RAPD)

Molecular typing (RAPD) was carried out using primer 1254 (CCGCAGCCAA) and primer 1290 (GTGGATGCGA) [17]. RAPD was carried out to determine whether each susceptible isolate and its resistant mutant belonged to the same strain, when the strips showed an identical profile between the susceptible isolate and its resistant mutant, after migration on an agarose gel.

DNA sequencing

Sequencing of the 23S rRNA gene which is associated with clarithromycin resistance was performed to determine the mutation type: A2142G or A2143G or A2143C, by sequencing (Applied Biosystems, version 3.1 or 1.1).

a)Culture in brucella broth

Isolates 3657-S/3657-R and 3695-S/3695-R were grown separately over a short period of time (48 hours) in macrolide-free brucella broth supplemented with 10% serum. Quantification was performed after 3, 6, 9, 25, 32 and 48 hours.

b)Competition in co-culture with eukaryotic cells

To assess the difference in the bacterial growth rate over a long time course, co-culture on the AGS gastric cell line was carried out for 30 or 40 days. Suspensions of similar opacity of the susceptible strain and its resistant mutant were used to infect the cells at a multiplicity of infection (MOI) of 2. Sequential passage of cells was carried out twice a week corresponding to 8 passage for 3695 isolate and 10 passage for 3657 isolates. The quantity of each isolate (susceptible and resistant) in CFU, was determined at the beginning of the experiment and at days 15 and 30 for strain 3695 and at days 15 and 40 for strain 3657, by culture on Mueller Hinton agar supplemented with 10% sheep blood and polyvitex with and without erythromycin and incubated in a microaerobic atmosphere. The CFU ratio between the susceptible strain and its resistant mutant was then recorded.

Then, Prepare a 0.5 MF suspension of each isolate in Brucella broth in a test tube;

Perform two dilutions (10⁻¹ and 10⁻²) in 15 ml flat-bottomed tubes: 4.5 ml of Brucella broth + 0.5 ml of bacterial suspension;Distribute 4.5 ml of the previously prepared Brucella broth (100 ml of broth + 5 ml of fetal calf serum) into 16 tubes (4 tubes for isolate 3657 S, 4 tubes for isolate 3657 R, 4 tubes for isolate 3695 S and 4 tubes for isolate 3695 R);Distribute 500 µl of bacterial suspension into the 8 tubes;Remove tube 1 (t0), culture it, and culture the other tubes with shaking in a microaerobic atmosphere;Remove the tubes at 3 hours, 6 hours, 9 hours, 12 hours, 24 hours, 36 hours and 48 hours and, from 1 ml of suspension, culture the pure suspension and the 10⁻³, 10⁻⁴, 10⁻⁵ and 10⁻⁶ dilutions in triplicate on MH agar + 10% sheep blood + Polyvitex (100 µl to spread with a sterile spreader);Also culture the uninoculated negative control tube;Dry the plates under a laminar flow hood;Incubate the plates in a microaerobic atmosphere for 5 to 6 days, then count the bacteria and calculate the ratio of resistant bacteria/sensitive bacteria.

The cell culture media were prepared by mixing the following reagents:

- 45 ml of DEMXII;

- 5 ml of fetal calf serum (serum D, see in the freezer);

- 50 µl of vancomycin (see in the freezer, microtubes labeled V);

- Filter this entire mixture with a 0.22 filter and syringe by screwing it into another 15 ml tube (remove the plunger before filtering).

Cell detachment, recovery, and flask preparation were performed as follows:

- Recover the medium contained in each flask and discard it in the waste container;

- Rinse the cells with PBS (10 ml), then recover all the supernatant and discard it in the waste container;

- Add 3 ml of trypsin; - Incubate at 37˚C for 5 minutes;

- Stop the trypsin action by adding 7 ml of medium;

- Transfer everything into a small 15 ml conical tube;

- Centrifuge (program 6, i.e., 1300 rpm for 3 min);

- Discard the supernatant;

- Resuspend the cells in 5 ml;

- Cell count: 15 µl of trypan blue + 15 µl of cells, take 10 µl and place in the counting chamber, the cell counter indicates: 1.13 × 10⁶ cells per ml, i.e., 5.65 × 10⁶ cells in 5 ml;

- Prepare 4 flasks (3 to be infected the next day and 1 negative control), by placing 1.13 million cells in each, i.e., 1 ml.

Cell infection with the 3657 S/3657R isolate pair

Preparation of a suspension with isolate 3657 S and another suspension with isolate

3657 R. Calibration of the suspensions to an OD of 1 using a spectrophotometer.

Calculation of the volume to inoculate into the flasks with an MOI of 1 (for 1.13 million cells), knowing that an OD of 1 corresponds to 2 × 10⁸ bacteria per 1 ml, which corresponds to a volume of 22 µl (for an MOI of 2).

Cell infection with the 3695 S/3695R isolate pair

Preparation of a suspension with isolate 3657 S and another suspension with isolate

3657 R. Calibration of the suspensions to an OD of 1 using a spectrophotometer.

Calculation of the volume to inoculate into the flasks with an MOI of 1 (for 1.13 million cells), knowing that an OD of 1 corresponds to 2 × 10⁸ bacteria per 1 ml, which corresponds to a volume of 22 µl (for an MOI of 2).

Cell passaging

The infected cells were passaged twice a week for at least 30 days (1 month) and 40 days, and new cells were infected with the previously infected cells.

a)Genome sequencing

DNA was extracted using the QIAmp DNA Mini Kit (Qiagen, Crawley, UK) according to the manufacturer’s instructions. Nucleic acid content was quantified and assessed with a Nanodrop spectrophotometer prior to normalization and sequencing. NexteraXT (v3 Technology, 250bp paired-end) libraries were prepared and high-throughput genome sequencing was performed using an Illumina MiSeqSystem benchtop sequencer (Illumina, San Diego, CA, USA). Short reads were assembled de novo using SPAdes (version 3.0.0) and were evaluated using QUAST [18].

b)Data base archiving,annotation and phylogeny

Contiguous genome sequences were imported and archived on the Bacterial Isolate Genome sequence database (BIGSdb) [19]. Putative coding sequences were identified for each assembled genome using the rapid annotation server RAST [20]. A pan-genome was constructed from all loci identified in at least one of the isolates used in this study using a reference pan-genome approach [21]. This derived list was used to interrogate each of the genomes analyzed in this study (BLASTN). Genes were defined as present by a match of greater than 70% identity over 50% or more of the locus length. Gene-by-gene alignments were exported to investigate single nucleotide polymorphism (SNP) variation. A neighbour-joining tree of the start and end isolates for both resistant and susceptible versions of the two strains was constructed using FastTree2 [22] and annotated with Evolview [23] according to the clarithromycin resistance profile and day of isolation. Individual SNPs in genes highlighted as potential hosts for compensatory mutations were analyzed from gene-by-gene alignments using Bioedit.

Each susceptible isolate and its respective clarithromycin resistant mutant were identified by culture and PCR and named 3657-S, 3657-R and 3695-S and 3695-R, respectively.

The MIC for both susceptible isolates was below the limit of 0.125 µg/ml and the 3695 resistant mutant MIC was higher than that of the 3657 resistant mutant (see Table 1).

The RAPD showed that in the two cases, the susceptible isolate and its resistant mutants had the same strain profile (see Figure 1).

Table 1. Clarithromycin minimal inhibitory concentration (MIC) measured in the 2 versions (labeled R for Resistant and S for Susceptible) of 2 clinical isolates (3657 and 3695).

S (Susceptible isolate), R (resistant isolate), M (molecular 1 Kb mass standard), C+ (external control, another H.pylori strain), C− (negative control).

Figure 1.Representative RAPD patterns of H.pylori 3695-S/3695-R and 3657-S/3657-R on 2% agarose gel.

Sequencing of the 23S rRNA gene of the resistant mutant showed an A2142G mutation in the 3695-R isolate and an A2143G mutation in the 3657-R isolate.

Figure 2. (a) Growth rate of H.pylori3657-S and 3657-R isolates in short term culture (R/S ratio at 48 hours = 7.2); (b) Growth rate of H.pylori3695-S and 3695-R isolates in short term culture (R/S ratio at 48 hours = 3.4).

Resistant mutants grew faster than the susceptible isolates in short term broth culture (48 hours) in both cases (3657 and 3695 strains), (see Figure 2(a) and Figure 2(b)).

For both pairs of isolates (susceptible and resistant), the growth of the resistant mutant at 15 days was better than that of its susceptible counterpart. However, at 30 or 40 days a difference was observed. The growth rate of the 3657-R mutant still remained higher than that of its susceptible counterpart while the contrary was observed for 3695 (Figure 3(a) and Figure 3(b)).

Figure 3. (a) Growth rate of H.pylori3695-S and 3695-R (R/S ratio at day15 = 4; R/S ratio in long term culture (day 30 = 0.1); (b) Growth rate of H.pylori3657-S and 3657-R (R/S ratio at day 15 = 1; R/S ratio in long term culture (day 40 = 1.7).

Figure 4. Circular cladogram view of a neighbour-joining tree built on a 1,508,595 bp alignment of the resistant and susceptible versions of strains 3657 and 3695.

The mean number of contigs for the 11 isolates tested was 50.5 giving rise to a mean total length of 1,648,591 bp. The pan genome included 1538 genes and the core genome alignment 1,508,595 bp. Resistant and susceptible isolates clustered closely on a phylogenic tree (Figure 4). Concerning the resistant isolate 3657-R, notable changes occurred in two genes: HP_0316 (hypothetical protein) and HP_1342 (putative outer membrane protein) between the start isolate (R0) and the isolate obtained after 40 days of cellular co-culture (R40). The alignments are presented in Table 2 and Table 3. Potential compensatory mutations may have occurred in these two genes. In contrast, no major changes in genes, no any significant mutation appeared in the resistant isolate 3695-R after 30 days of co-culture (Table 4 to Table 5).

Table 2. Hp 1342 alignments: strain 3657 (modifications corresponding to putative outer membrane protein).

Table 3. Hp 0316 alignments: strain 3657 (corresponding to hypothetical protein).

Table 4. Hp 0316 alignments: strain 3695.

Table 5. Hp 1342 alignments: strain 3695.