Evaluation Of Combination Effects Of Antibiotics Against Helicobacter Pylori Clinical Isolates In Vitro

Background&Objective: Helicobacter pylori(H.pylori) infection has been associated with upper gastrointestinal tract disorders, the elementary treatment strategy is to select antimicrobial spectrum involved medicine. It is well known that any kind of antibacterial agents can achieve the purpose when be independently used, two kinds of antibiotics in combination thereby are the inevitable choice. Combination will generate diverse effects, and a reasonable optimal combination programme will enhance the therapeutic efficiency and narrow the mutant selection window(MSW) to diminish the possibility of strains resistant mutation. Current expert opinions for the treatment of H.pylori have contained 8 kinds of antibiotic combinations which mainly based on resistance rate and eradication rate, but the combination effects cannot analyze and judgement owing to the sparse relative research and absence of reliable proof. In addition, other existential value antibiotic combinations untouched in consensus will be taken into consideration as well. To observing the antibacterial effects of 6 latest consensus recommended antimicrobial agents(amoxicilin-AM, clarithromycin-CH, metronidazole-MZ, levofloxacin-LE, furazolidone-FR or tetracycline-TC) used alone and combined with each other in vitro and evaluating the combination effects, the study was designed to provide experimental foundation for the selection of sensitive antimicrobial agents and efficient combinations to against drug-resistant H.pylori and explore the existential value of other free antibiotic combinations simultaneously.Methods: 32 strains of drug-resistant H.pylori were selected from retained clinical isolates, the drug sensitivity of which had been detected by using E-test or K-B method. ATCC® 43504 was chosen as quality control strains. Bacterial susceptibility testing was carried out by using the checkerboard method and the dilution agar method according to the CLSI performance standards. Then, the minimal inhibition concentration(MIC) of 6 antimicrobial agents used alone and combined with each other were obtained, and the fractional inhibitory concentration indexs(FICI) were calculated to evaluate combination effects. When FICI≤0.5 was defined as synergism,0.5<FICI≤1 as accumulation, 1<FICI≤2 as independence and FICI>2 as antagonism.Results: 1. For quality control strains, the MIC of AM, CH, MZ, LE, FR, TC were 0.031 μg/m L, 0.125μg/m L, 128μg/m L, 0.125μg/m L, 0.063μg/m L, 0.125μg/m L separately. 2. For 32 clinical isolates, the range of MIC of AM, CH, MZ, LE, FR, TC were 0.063~4μg/m L, 0.031~256μg/m L, 2~256μg/m L, 0.031~32μg/m L, 0.031~0.063μg/m L, 0.031~2μg/m L. There were 3 AM-resistant strains, 17 CH-resistant, 30 MZresistant, 20 LE-resistant, 0 FR-resistant, 2 TC- resistant and 9 strains(28.1%) for single antibiotic(s), 10(31.3%) for double and 13(40.6%) for multiple among them in total. 3. The FICI in 15 groups were showed as follow: ①AM+CH: FICI≤0.5(18.8%), 0.5<FICI≤1(71.9%), 1<FICI≤2(9.3%); ②AM+MZ: FICI≤0.5(84.4%), 0.5<FICI≤1(15.6%); ③AM+LE: FICI≤0.5(93.9%), 0.5<FICI≤1(6.1%); ④AM+FR: FICI≤0.5(9.3%), 0.5<FICI≤1(59.4%), 1<FICI≤2(31.3%); ⑤AM+TC: 1<FICI≤2(68.8%), FICI>2(31.2%); ⑥CH+MZ: FICI≤0.5(25.0%), 0.5<FICI≤1(75.0%); ⑦CH+LE: 1<FICI≤2(46.9%), FICI>2(53.1%); ⑧CH+FR: 0.5<FICI≤1(78.1%), 1<FICI≤2(21.9%); ⑨CH+TC: 0.5<FICI≤1(87.5%), 1<FICI ≤2(12.5%); ⑩MZ+LE: FICI≤0.5(18.7%), 0.5<FICI≤1(50.0%), 1<FICI≤2(31.3%); ?MZ+FR: 0.5<FICI≤1(68.8%), 1<FICI≤2(31.2%); ?MZ+TC: FICI≤0.5(25.0%), 0.5<FICI≤1(75.0%); ?LE+FR:0.5<FICI≤1(62.5%), 1<FICI≤2(21.9%), FICI>2(15.6%); ?LE+TC: FICI≤0.5(56.3%), 0.5<FICI≤1(43.7%); ?FR+TC: FICI≤0.5(15.6%), 0.5<FICI ≤1(62.5%), 1<FICI≤2(21.9%).Conclusions: In vitro, combination effects of AM+MZ/LE were mainly synergistic, of AM+CH/FR, CH+MZ/FR/TC, MZ+LE/FR/TC, LE+FR/TC, FR+TC were additive. Whlie AM+TC primarily showed indifferent, antagonistic action was showed respectively in the combinations of AM+TC(31.2%), CH+LE(53.1%) and LE+FR(15.6%). Attention should pay to the antagonistic effect will be brought in when antibacterial agents were combined inappropriately. Distinctly, consideration must be given to both the antibiotic-resistance problem of H.pylori and the rational antimicrobial combinations.