Nontypeable Haemophilus influenzae isolates from the lower respiratory tract in Western Sichuan, China: MIC value of ofloxacin and Mutation characteristics of Quinolones Resistance determining region gene


 Background: Fluoroquinolones are one of the most widely used antibiotics in the treatment of respiratory tract infections, and the mechanism of resistance to fluoroquinolones is considered to be related to the amino acid substitution of gyrA, gyrB, parC, and parE, the Quinolone Resistance-Determining Regions (QRDRs) of DNA cyclase type II topoisomerase and IV topoisomerase. The purpose of this study was to explore the molecular mechanism of quinolone resistance of Nontypeable Haemophilus influenzae(NTHi ) isolates and analyze the mutation law of the QRDRs gene. Results: MIC value of ofloxacin of 280 NTHi isolates from lower respiratory tract secretions in children group during 2003~2004 and in whole age group during 2013~2014 in Western Sichuan, China were monitored and the amino acid sequences of gyrA, gyrB, parC and parE gene in QRDRs were detected. The resistance rate of ofloxacin in adult group was 1.92% (n=52), while the NTHi strains with ofloxacin MIC value≥0.5 showed an upward trend in all age groups. No ofloxacin-resistant strains were found in 57 NTHi strains isolated from the children patient. Four amino acid substitutions were found in GyrA genes, four in GyrB, three in parC and nine in parE genes. The results showed that different amino acid replacement patterns of the gyrA , gyrB, parC and parE gene had different effects on ofloxacin MIC values. Conclusions: Ser-84-leu and asp-88-tyr/asn mutation of the gyrA, ser-84-lys/ile and ser -133-ala mutations of the parC and ala-400-val variation of the gyrB were the main factors leading to the increase of MIC value of ofloxacin of NTHi strains in Western Sichuan, China. It can be predicted that with the increase of quinolones exposure, the susceptibility of isolates from children to quinolones will be further reduced.

pediatrics in China, our early studies found that H. influenzae, which is resistant to ciprofloxacin, were isolated from the respiratory tract of children and newborns [2]. In order to explore the molecular mechanism of fluoroquinolones resistance and the genetic regularity of drug resistance genes, we monitored the susceptibility to ofloxacin of NTHi isolates from lower respiratory tract secretions from  Table and in Table 2.     The distribution of the strains with 8 allelic mutations in the three age groups is shown in S4 table (results of χ2 test or Fisher exact probability method). The distribution trend of the above QRDRs allele mutations in the three age groups is shown in Fig. 2. The age group distribution of ser-84-leu and asp-88-tyr/asn as well as ala-134-val mutation in gyrA sequence was significantly different (p = 0.013, 0.034 and 0.010, respectively), but the other five loci had no statistical significance, p > 0.05.

Discussion
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][21][22][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 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, 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.

Conclusions
Ser-84-leu and asp-88-tyr/asn mutation of the gyrA, ser-84-lys/ile and ser − 133-ala mutations of the parC and ala-400-val variation of the gyrB were the main factors leading to the increase of MIC value of ofloxacin of NTHi strains in Western Sichuan, China. It can be predicted that with the increase of quinolones exposure, the susceptibility of isolates from children to quinolones will be further reduced.   [5,6]. Outer membrane protein P6 is a member of the class of outer membrane proteins known as peptidoglycan-associated lipoproteins, which are highly conserved among H.

Strain source
influenzae isolates [7]. The fucK gene has higher specificity for identification of H. influenzae [8].

Data analysis
The enumeration data were expressed as percentage (%). There was a significant difference in χ2 or Fisher exact probability test between the two groups by using Statistical Software R (v3.3.1), and the difference was statistically significant with p < 0.05. The MIC values of the two groups were compared by Rank Sum test. The median and quartile intervals were used to describe the center and the discrete trend, and p < 0.05 is statistically significant for the difference.Two independent sample rank sum test (Mann-Whitney u test) and Multivariate Ordered Logistic Regression Analysis with spss20.0 Statistical Software for the data satisfying the condition. The independent variable is the mutation point of the gyrA gene, gyrB gene, parC gene and parE gene, respectively; and the dependent variable is the value of ofloxacin MIC value, in Multivariate Ordered Logistic Regression Analysis. The criteria is 0.05, exclusion criteria is 0.10; p < 0.05 is statistically significant for the difference. Trend of partial QRDRs variation in the past decade.

Figure 2
Comparison of partial QDRDs variation in different age groups.

Supplementary Files
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