Ofloxacin MIC values and Mutation characteristics of Quinolones resistance determining region gene of Haemophilus influenzae isolates from the lower respiratory tract in Western Sichuan, China: Comparative study of children and adults

Abstract Background: In order to further investigate the trend of quinolone resistance and the gene variation characteristics of quinolone resistance determining region of non-typeable Haemophilus influenzae (NTHi) isolates from Chinese children's respiratory tract, We tracked the isolates of children in western Sichuan of China for ten years and compared them with adult groups in the same period.Method: We monitored the MIC value of ofloxacin of NTHi isolates (n=280) from lower respiratory tract secretions in children group (n=57) during 2003~2004 and in whole age group (n=223) during 2013~2014 in Western Sichuan, China. The amino acid sequences of QRDRs, gyrA, gyrB, parC and parE, were detected; and the relationship between amino acid substitutions (AAS) of QRDRs and ofloxacin MIC value was analyzed. At the same time, the mutation trend of QRDRs gene in this region during the past ten years was compared analyzed.Results: 1. All the strains (n=280) of H. influenzae included in the study were NTHi. No ofloxacin-resistant strains were found in 57 NTHi isolated from the children patient during 2003~2004. While strains with the minimum inhibitory concentration (MIC) value ≥ 0.5 showed an upward trend in all age groups during 2013~2014. 2. The AAS are S84L, D88Y/N, A134V and E142K in gyrA, G399E, A400V, E469D and T472I in gyrB. S84R/I, S133A and N138S in parC and D364Y, A369T, R378C, A383T, G405S, D420N, A426V, V466M and S474N in parE. 3. Compared to the non-mutation group, the change of ofloxacin MIC values of the strains with S84L, D88Y/N and A134V in gyrA, A400V in gyrB and S84R/I , S133A in parC showed statistical significance (p≤0.05 or approximate p=0.05); And the results of ordered multi-classification logistic analysis showed that the AAS gyrA-S84L (OR=139.824, 95% CI=55.730~350.811, p＜0.001), gyrA-D88Y/N (OR=28.950, 95% CI=8.432~99.395, p＜0.001), parC-S84R/I (OR=102.789, 95% CI=31.851~331.713, p＜0.001), parC-S133A (OR=1.872, 95% CI=1.023~3.426, p=0.042) were risk factors for the increase of ofloxacin MIC value, respectively; the AAS gyrB-A400V (OR=0.517, 95% CI=0.322~0.831, p=0.006) was the factor affecting the decrease of ofloxacin MIC value. The gyrA’s S84L, gyrA’s D88Y/N, gyrB’s A400V, parC’s S84R/I and parC’s S133A mutations were the main factors affecting the MIC value of ofloxacin of NTHi isolates in Western Sichuan, China, respectively. 4. The age group distribution of mutations S84L, D88Y/N and A134V in gyrA were significantly different (p=0.013, 0.034 and 0.010, respectively). The key mutation of multiple QRDRs of NTHi isolates in children in low age group is increasing rapidly. Along with increase of the mutation rate of S84L in gyrA and A400V in gyrB of isolates from 0~3 yrs old group (X2=6.089, p=0.014; X2=25.181, p<0.001), the ofloxacin MIC value increasing was statistically significant in the past ten years (p=0.022).