Broad spectrum resistance in Helicobacter pylori isolated from gastric biopsies of patients with dyspepsia in Cameroon and efflux-mediated multiresistance detection in MDR isolates

This was a cross sectional study carried out at the gastroenterology Department of Laquintinie Hospital and General Hospital of Douala-Cameroon, from April 2013 to June 2015. All patients aged 15 to 90 years old, either sex, attending the Gastroenterology Department at the selected health centers with gastritis, gastric and duodenal ulcer undergoing endoscopy were recruited. Exclusion criteria were, (1) Non-cooperative patients who refused to give their consent or to participate to the study; (2) patients with a history of H. pylori eradication treatment; (3) patients with a history of antibiotics consumption within the last fourth weeks. Gastric biopsies were collected from the enrolled patients, ground and about two drops of homogenates were inoculated into supplemented Columbia Agar (Columbia Agar + 5% (v/v) lacked horse blood and 1% (v/v) Vitox (CD-Vitox)). The inoculated culture media were incubated at 37 °C under microaerophilic conditions (CampyGen gas pack) for 7 days. Isolates that exhibited Gram negative curved rods on Gram stain reaction and were positive for catalase, oxidase and urease tests were considered as H. pylori [27, 28]. Confirmed isolates were suspended in eppendorf tubes containing Brain Heart Infusion broth supplemented with 5% horse serum (BHI-serum) with 30% glycerol and stored at − 80 °C until future use. The study was approved by both local ethical committee (Approval N⁰ 425/AR/MINSANTE/HLD/SCM/CR) and the national institutional Review Board, the National Ethical Committee on human health research in Cameroon (ethical clearance N⁰ 2014/03/425/L/CNESRH/SP).A standard control strain NCTC 11638 was also used to confirm the identification and for antimicrobial assays.

One hundred and forty (140) H. pylori isolates were recovered from gastric biopsies of eligible patients and subjected to antimicrobial assays in this study. All isolates were removed from storage at − 80 °C and subcultured on supplemented Columbia Agar (Columbia Agar + 5% (v/v) lacked horse blood and 1% (v/v) Vitox (CD-Vitox)). Subcultures were incubated at 37 °C under microaerophilic conditions (CampyGen gas pack) for 3 days and for two passages to ensure reliable growth. Gram staining, rapid urease and the catalase/oxydase tests were performed to confirm the identification [28, 29].

Antibiotic susceptibility studies were performed by disc diffusion (Kirby–Bauer) method according to Clinical Laboratory Standards Institute (CLSI, 2015) [29]. The following antibiotic disks were tested: amoxicillin (10 μg), co-amoxiclav (30 μg), ampicillin (10 μg), penicillin (10 U), imipenem (10 μg), metronidazole (5 μg), rifabutin (5 μg), erythromycin (15 μg), clarithromycin (15 μg), azithromycin (15 μg), levofloxacin (5 μg), ciprofloxacin (30 μg), norfloxacin (30 μg), tetracycline (30 μg), doxycycline (30 μg) and minocycline (30 μg) (Biomaxima). H. pylori inocula prepared at McFarlands turbidity standard 3 (6 × 10 ⁸ CFU/ml) by suspending 48-h colonies in 2 ml-sterile normal saline was used to seed each prepared and dried supplemented Columbia agar plate. The discs were arranged and firmly pressed on the agar surface of each seeded plate. The plates were incubated under microaerophilic conditions at 37 °C for 48 to 72 h. The microaerophilic conditions were generated using CampyGen in an air-tight anaerobic jar (Oxoid). Zone sizes were measured and the isolates were classified as sensitive or resistant according to the interpretative criteria for CLSI guidelines (2012) and CASFM (2017) [30, 31]. The isolate exhibited a multidrug resistance phenotype were also noticed. The experiment was performed in triplicate and the mean zone diameters of inhibition recorded for each antibiotic.

In order to determine a possible mechanisms involved in resistance pattern of the tested clinical isolate against antibiotics used, overexpression of MDR efflux pumps in the isolated H. pylori strains was investigated using Phe-Arg-naphthylamide (PAßN). Thirty two (32) isolates which elicited triple or quadruple drug-multi resistance pattern to the tested antibiotics according to the above test were selected. Selected MDR isolates were tested for their susceptibilities to antibiotics amoxicillin (AMO), ampicillin (AMP), clarithromycin (CLR), erythromycin (ERY), metronidazole (MET), tetracycline (TET) and ciprofloxacin (CIP) alone and then in the presence of PAßN at a final concentration of 20 μg/ml as described previously [32]. The MICs were carried out by the INT broth microdilution method [33, 34] using Brain Heart Infusion broth supplemented with 5% horse serum (BHI-serum) and various concentrations of antibiotics. Two fold dilutions of each selected antibiotics were prepared in the test wells in BHI -serum. The ranges of antibiotic concentrations that were used in this study were as follows: amoxicillin (AMO, 0.03125–128 μg/ml), ampicillin (AMP, 0.03125–128 μg/ml), Clarithromycin (CLR, 0.0625–16 μg/ml), erythromycin (ERY, 0.125–32 μg/ml), metronidazole (MET, 0.5–128 μg/ml) and doxycycline (DOX, 0.0625–32 μg/ml). All antimicrobial agents were purchased from Sigma-Aldrich, Germany. The selected MDR H. pylori isolates were removed from freezer, thawed and subcultured on supplemented Columbia Agar without antibiotics and incubated under microaerophilic conditions at 37 °C for 3 days. After a lawn of growth appeared, MDR bacterial colonies were suspended in sterile saline at a density equivalent to 3 McFarland’s standard. One hundred microliter of each prepared inoculum was added to 100 μl of the antibiotic-containing culture medium or antibiotic+PAßN-containing culture medium. Control wells were prepared with culture medium and MDR bacterial suspension, and broth only. The plates were covered with a sterile plate sealer; the contents of the wells were mixed with a shaker and incubated for 3 days at 37∘C under microaerophilic conditions. After incubation, 40 µl of 0.2 mg/ml INT was added per well and incubated at 37∘C for 30 min. Living bacteria reduced the yellow dye to pink. The MIC value of antibiotic defined as its lowest concentration which completely inhibited visible bacterial growth at 37 °C after 72 h was determined in the presence and in the absence of efflux pump inhibitor. Each MIC was determined in triplicate and the mean values were recorded. Then, the fractional inhibitory concentration (FIC) of each combination was calculated as the ratio of MIC of antibiotic with efflux pump inhibitor versus MIC of antibiotic alone. Resistance was defined in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines [35]: amoxicillin, ampicillin (MIC > 0.125 μg/ml), clarithromycin (MIC > 0.5 μg/ml), erythromycin (MIC > 1 μg/ml), tetracycline, doxycycline (MIC > 1 μg/ml); metronidazole (MIC > 4 μg/ml) [36].

In this study, the susceptibility of 140 H. pylori clinical isolates was evaluated against 16 antibiotics belonged to six class and they were classified according to their resistance profile to the tested drugs (Tables 1 and 2, Fig. 1). None of the tested strains elicited a 100% susceptibility to the overall antibiotics used. However, they did not showed any resistance to rifabutin, imipenem, azithromycin, ciprofloxacin, norfloxacin and levofloxacin (Table 2). Resistance rates recorded against the tested antibiotics were as follows: highest resistance rate (100 to 97.85%) to metronidazole and penicillin group (ampicillin, penicillin, co-amoxiclav, amoxicillin); 47.85 and 13.57% to erythromycin and clarithromycin respectively. Antibiotic from tetracyclines (tetracycline: 2.86%, doxycycline: 5% and minocycline: 0.71%) were slightly affected by resistance. Since there was a high disparity between the resistance rate of imipenem (0%) and that of the other β lactam antibiotics tested (ampicillin: 100%, penicillin: 100%, co-amoxiclav: 100% and amoxicillin: 97.14%), this class were divided in penicillin and carbopemems subgroups, and the resistance prevalence of this two subgroups were not joined in statistical analysis. So, the peak of resistance was detected with metronidazole (imidazoles class) and penicillin’s subgroup with respective resistance rate of 99.28 and 97.85%, followed by macrolides with 20.47%. However, none of the tested strains were resistance to azithromycin one of the three macrolides tested (Fig. 1). Rifabutin, the only antibiotic of the rifamycine class used and all the fluoroquinolone tested showed a 100% activity against the tested isolates.

Out of the 140 H. pylori isolates tested, 98 elicited a multiple resistance pattern, given the overall multidrug resistance rate of 70% (98/140). The incriminated antibiotics classes were imidazole, penicillin, macrolides and tetracyclines. Table 3 summarizes the multi-drug resistant pattern of tested H. pylori isolates; 68 (48.57%) elicited double, 22 (15.71%) triple and 8 (5.71%) quadruple drugs resistance. Double resistance were more frequent with metronidazole and amoxicillin (MET^R/AMO^R) which represented 86.76% (59/68) of this type of multi-drugs resistance pattern, followed by MET^R/CLR^R, MET^R/TET^R and CLR^R/AMO^R. As regards triple drug resistance, metronidazole, clarithromycin and amoxicillin (MET^R/CLR^R/AMO^R) were more concerned and represented 95.45% (21/22) of this type of multidrug resistance pattern. Quadruple drugs resistance was the maximum and the less multi-drugs resistance pattern detected and metronidazole, tetracycline, clarithromycin and amoxicillin were combined.

As possible mechanisms involved in resistance pattern of the tested clinical isolates against antibiotics used, overexpression of MDR efflux pumps in the isolated H. pylori strains was investigated using Phe-Arg-naphthylamide (PAßN). The susceptibility to antibiotics of selected MDR isolates (32) was evaluated in the presence and absence of the PAßN. The selected antibiotics were ampicillin, amoxicillin, metronidazole, erythromycin, clarithromycin and doxycycline to which the tested isolate displayed a resistance prevalence of 100, 97.14, 97.85, 47.85, 13.57 and 5% respectively. Table 4 summarizes the susceptibility of the selected MDR H. pylori isolate to antibiotics in the absence and presence of PAβN. The activity spectrum of amoxicillin and ampicillin was not affected in the presence of the efflux pumps inhibitor. However, our results showed that this inhibitor (PAßN) have improved the activity of 67% (4/6) of the antibiotics tested (that is metronidazole, doxycycline, clarithromycin and erythromycin). The spectrum of activity recorded with metronidazole, doxycycline, clarithromycin and erythromycin against the tested MDR H. pylori strains ranged in the absence to the presence of the inhibitor from 0 to 34.37%; from 40 to 100%; from 0 to 100% and from 6.90 to 100% respectively (Table 4).

An increase in potency of 128 and 64-fold was observed with metronidazole in 3.125% (1/32) of the selected MDR isolates, that of clarithromycin was 64 and 40-fold in 40% (2/5) of the selected isolates, 32 to 40-fold with erythromycin in 34.48% (10/29) of the tested isolates and 8-fold increase in potency with doxycycline in 60% (3/5) of the MDR isolates tested (Table 4).