Through an all age group epidemiological survey in 2013 ~ 2014, we found that H. influenzae isolated from sputum from patients with lower respiratory tract infection in Western Sichuan Province were all non capsular strains or called NTHi isolates(NTHi)[3, 4]. Ofloxacin resistance rate was 1.98% (2/101) in the group of ≥ 18 years old[4]. Although no resistant strain was found in the 0 ~ 17 years old group[3], the result of the more accurate broth drug sensitivity test showed that the strains of ofloxacin MIC ≥ 0.5 showed an increasing trend in all age groups. Indeed, H. influenzae isolated from all age groups is becoming less susceptible to ofloxacin. Our findings are very similar to the results of a simultaneous study by shoji S et al in Japan[10]. Unlike in China, tofloxacin (tosufloxacin) as one kind of fluoroquinolones has been approved for use in pediatric patients in Japan in 2010[11]. The reason why the strains of ofloxacin MIC ≥ 1 appeared in 0 ~ 3 years old patients and the susceptibility to ofloxacin was decreased in 0 ~ 17 years old patients in China should be paid more attention to. Since 1993, when quinolone-resistant strains were first reported [12], the strains with reduced susceptibility to quinolones have been found in elderly patients in many countries and regions[13, 14, 20–23]. Because a group of fluoroquinolones, such as tosufloxacin, levofloxacin, moxifloxacin, garenoxacin and sitafloxacin, etc., have an excellent transfer rate to the lungs and show a strong antibacterial activity against most community-acquired pneumonia-causing pathogens, including S. pneumoniae, H. influenzae and Mycoplasma pneumoniae[12], they are called as ‘respiratory quinolones’and are used as a first-line drug to treatment for adults with community-acquired pneumonia[15]. The widespread use of fluoroquinolones in adult patients may induce the emergence of more drug-resistant strains, but it does not explain why H. influenzae isolates with reduced ofloxacin sensitivity have been found more frequently in Chinese children. So the genetic pattern of quinolone-resistant strains isolated from Chinese children is worthy of further study.
The mechanism of action of the quinolones involves the disruption of DNA replication of type II topoisomerase[16]. Type II topoisomerases are currently recognized to include DNA gyrase, which is responsible for the formation and elimination of supercoiled structures in DNA strands, and topoisomerase IV, which cuts and re-ligates tangled DNA during DNA replication[16]. Each of these enzymes is composed of two dimers of subunit types A and B, which together form a tetramer. DNA gyrase is composed of gyrA and gyrB, topoisomerase IV comprises parC and parE [17]. The subunit A (gyrA and parC) possesses DNA cutting and ligating activity, the subunit B (gyrB and parE) possesses ATPase activity[18]. Quinolones bind to the exposed double-stranded DNA and form the DNA-DNA gyraseequinolone antibiotic complex, thereby preventing the re-ligation of DNA[19]. Substitutions of amino acids in each enzyme lead to the inhibition of these formations[19]. In particular, mutations in the QRDRs are closely related to resistance[19]. In particular, mutations in the QRDRs are closely related to resistance. Mutations in the QRDRs of H. influenzae have been demonstrated to occur in a stepwise manner with an increasing number of mutations yielding higher quinolone MIC[20–24]. A sequence of nucleotides associated with quinolones resistance in the gyrA and parC gene sequences is known as the QRDRs. Among them, the parC region of the gyrA gene was composed of the 202 ~ 531 base at the fifth terminal of the gyrA gene (that is, the base encoding the 68 ~ 177 amino acid residues of the gyrA) and the 152 ~ 456 base at the terminal of the parC gene (that is, the amino acid residue at the 51 ~ 152nd position of the parC). Previously, quinolone-resistant H. influenzae with substitutions at ser-84 and asp-88 in gyrA, as well as at gly-82, ser-84, glu-88 in parC have been reported[25, 26]. Kurt F et al [27] reported that in addition to the ser-84-leu and asp-88-asn in gyrA and the ser-84-ile in parC, the molecular characteristics of high levels of H. influenzae resistant to ciprofloxacin in southern Denmark also included K20R, asp-356-ala/thr-356-ala and met-481-ile mutation of parC and glu-151-lys, ile-159-ala, asp-420-asn and ser-599-ala mutation of parE. Hisashi S et al. [28] showed that five amino acid substitutions in gyrA (at ser-84 and asp-88) and parC (at gly-82, ser-84 and glu-88) were found to be closely related to the MIC by genetic transformation experiments. It was also observed that the degree of resistance is related to the number of the mutations[28].
In addition to ser-84-leu, asp-88-tyr/asn mutation, the gyrA sequence of ala-134-val and glu-142-lys mutation was also observed. In addition to ser-84-lys/ile mutation, ser-133-ala and asn-138-ser mutation were also observed in the QRDRs sequence of parC. We also observed the amino acid substitution of the gyrB, gly-399-glu, ala-400-val, glu-469-asp and thr-472-ile, which were rarely reported in Asia before. The amino acid substitution of 9 sites was also found in the parE sequence, including asp-364- tyr, ala-369-thr, lys-378-cys, ala-383-thr, cys-405-ser, asp-420-asn, ala-426-val, val-466-met and ser-474-asn. We tried to analyze the effect of QRDRs amino acid substitution on the MIC value of ofloxacin by statistical method (orderly multiple classifications logistic regression analysis), and the results showed that the ser-84-leu and asp-88-tyr/asn mutation in gyrA sequence were the main factors affecting the MIC value of ofloxacin (p < 0.001); and the effect of the variation on MIC value of ofloxacin was 139.824 times and 28.950 times of that of no variation, respectively. Ser-84-lys/ile and ser-133-ala, the amino acid substitution of parE sequence, were the main factors affecting the MIC value of ofloxacin (p < 0.001 and p < 0.042); and the effects of variation on MIC value of ofloxacin were 102.789 times and 1.872 times of that of no variation, respectively. Ala-400-val, the amino acid substitution of gyrB sequence, was the main factor affecting the MIC value of ofloxacin (p = 0.006); and the effect of variation on MIC value of ofloxacin was 0.517 times of that of unmutated strains. In addition, the results of Mann-Whitney U test showed that the ala-134-val and glu-142-lys variations of gyrA sequence and asn-138-ser variation of parC sequence were significantly different from those of unmutated group ofloxacin MIC, p ≤ 0.05. However, the further ordered multiple classifications logistic regression analysis showed that the effect of the above three-locus variations on the MIC value of the strain was not statistically significant (p = 0.860, 0.783, and 0.200, respectively). Our study also found that the amino acid substitution of parE sequence was complex, but the further statistical analysis indicated that the variation of parE sequence had no statistical significance on the MIC value of ofloxacin. Previously, Hisashi S et al.[28] verified the ser-84-leu, asp-88-tyr/asn, glu-142-lys mutation of gyrA and the ser-84-lys/ile and asn-138-ser mutation of parC by means of genetic transformation experiments, and the results were consistent with the results of this study. However, Hisashi S et al found that 8 strains, containing the met-121-leu and glu-142-lys mutations of gyrA, the ala-400-val mutations of gyrB, the ser-133-ala and asn-138-ser mutations of parC, the val-466-metmutation of parE, showed a low range of moxifloxacin MIC (0.015–0.06 mg/ml) [28], which was not exactly consistent with our observation. According to the statistical results, we inferred that the QRDRs mutation sites affecting the MIC value of ofloxacin were gyrA's ser-84-leu (139.824 times), parC's ser-84-lys/ile (102.789 times), gyrA's ser-133-ala (28.950 times), parC's ser-133-ala (1.872 times) and gyrB's ala-400-val (0.517 times), respectively. while in the same period a further study on the mechanism of beta-lactam resistancer of the NTHi strain, the results showed that different amino acid replacement patterns of the ftsI gene had different effects on the MIC values[29]. However, statistical analysis is not a substitute for further genetic transformation experiments and Ka/Ks evolutionary analysis, Ka/Ks is the ratio of the number of nonsynonymous substitutions per nonsynonymous site (Ka) to the number of synonymous substitutions per synonymous site (Ks), to clarify the real role of amino acid substitution in the mechanism of quinolone resistance emerging in the study and clarify the interrelationship between these amino acid substitutions.
In the past ten years, the strains with ser-84-leu mutation of gyrA sequence that led to the significant increase of the MIC value of ofloxacin, while the strains with ala-400-val mutation of gyrB sequence also increased significantly in western Sichuan increased significantly. It is suggested that not only a key mutation in the nucleic acid sequence in QRDRs of type II topoisomerase, but also the change of ATPase activity may be involved in the quinolone- resistance mechanism of H. influenzae strain.
Although the strains with the ser-84-leu,asp-88-tyr/asn and ala-134-val mutation, which were isolated from the 0 ~ 3yrs-old group and from the 4-6yrs-old group, were still at a low level, however, the strains with the glu-142-lys mutation of gyrA sequence, with ala-400-val mutation of gyrB sequence, and with the ser-84-lys/ile and ser-133-ala as well as asn-138-ser mutation, were similar to those of the adult group. It can be predicted that with the increase of quinolones exposure, the susceptibility of isolates from children to quinolones will be further reduced.