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      Molecular Epidemiology and Antimicrobial Resistance of Haemophilus influenzae in Adult Patients in Shanghai, China

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          Abstract

          Background: The serotype and antimicrobial resistance of Haemophilus influenzae in adult patients have changed due to the application of antimicrobials and H. influenzae type b (Hib) vaccine worldwide. However, the epidemiologic characteristics of H. influenzae in Shanghai are still unavailable.

          Objective: To determine the serotype distribution, antimicrobial resistance and multilocus sequence type (MLST) of H. influenzae in adult patients in Shanghai.

          Methods: A total of 51 clinical isolates from adult patients were consecutively collected. Serotypes were determined according to specific capsule gene, bexA, amplified by PCR. Antimicrobial susceptibility test was carried out by the broth microdilution method. β-lactamase production was detected by cefinase disk and the ftsI gene were amplified and sequenced to determine the penicillin binding protein 3 (PBP3) mutation. Molecular epidemiology was performed by MLST analyses.

          Results: All isolates studied were nontypeable H. influenzae (NTHi) and three of them (5.88%) caused invasive infection. The resistant rates of ampicillin and trimethoprim/sulfamethoxazole were both 45.10%. One third of these isolates produced TEM-1 type β-lactamase and 11.76% were β-lactamase negative ampicillin resistant strains (BLNAR). The PBP3 mutation was detected in 74.51% of the isolates, of which 12 belonged to group III. A total of 36 sequence types (STs) were identified among all isolates. Four isolates of ST103 (7.84%) all produced β-lactamase without mutation of PBP3.

          Conclusion: H. influenzae infections among adults in Shanghai are predominately caused by NTHi with genetic diversity among adult patients. The prevalence of both β-lactamase production and PBP3 mutation may contribute to high ampicillin resistance rate in Shanghai.

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          Most cited references29

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          PCR for capsular typing of Haemophilus influenzae.

          A PCR method for the unequivocal assignment of Haemophilus influenzae capsular type (types a to f) was developed. PCR primers were designed from capsule type-specific DNA sequences cloned from the capsular gene cluster of each of the six capsular types. PCR product was amplified only from the capsular type for which the primers were designed. Product was confirmed by using either an internal oligonucleotide or restriction endonuclease digestion. A total of 172 H. influenzae strains of known capsular type (determined genetically) comprising all capsular types and noncapsulate strains were tested by PCR capsular typing. In all cases the PCR capsular type corresponded to the capsular genotype determined by restriction fragment length polymorphism analysis of the cap region. When used in conjunction with PCR primers derived from the capsular gene bexA, capsulate, noncapsulate, and capsule-deficient type b mutant strains could be differentiated. PCR capsular typing overcomes the problems of cross-reaction and autoagglutination associated with the serotyping of H. influenzae strains. The rapid and unequivocal capsular typing method that is described will be particularly important for typing invasive H. influenzae strains isolated from recipients of H. influenzae type b vaccine.
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            Invasive Disease Caused by Nontypeable Haemophilus influenzae

            These infections are emerging worldwide, especially in young children and the elderly.
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              Molecular Surveillance of True Nontypeable Haemophilus influenzae: An Evaluation of PCR Screening Assays

              Background Unambiguous identification of nontypeable Haemophilus influenzae (NTHi) is not possible by conventional microbiology. Molecular characterisation of phenotypically defined NTHi isolates suggests that up to 40% are Haemophilus haemolyticus (Hh); however, the genetic similarity of NTHi and Hh limits the power of simple molecular techniques such as PCR for species discrimination. Methodology/Principal Findings Here we assess the ability of previously published and novel PCR-based assays to identify true NTHi. Sixty phenotypic NTHi isolates, classified by a dual 16S rRNA gene PCR algorithm as NTHi (n = 22), Hh (n = 27) or equivocal (n = 11), were further characterised by sequencing of the 16S rRNA and recA genes then interrogated by PCR-based assays targeting the omp P2, omp P6, lgtC, hpd, 16S rRNA, fucK and iga genes. The sequencing data and PCR results were used to define NTHi for this study. Two hpd real time PCR assays (hpd#1 and hpd#3) and the conventional iga PCR assay were equally efficient at differentiating study-defined NTHi from Hh, each with a receiver operator characteristic curve area of 0.90 [0.83; 0.98]. The hpd#1 and hpd#3 assays were completely specific against a panel of common respiratory bacteria, unlike the iga PCR, and the hpd#3 assay was able to detect below 10 copies per reaction. Conclusions/Significance Our data suggest an evolutionary continuum between NTHi and Hh and therefore no single gene target could completely differentiate NTHi from Hh. The hpd#3 real time PCR assay proved to be the superior method for discrimination of NTHi from closely related Haemophilus species with the added potential for quantification of H. influenzae directly from specimens. We suggest the hpd#3 assay would be suitable for routine NTHi surveillance and to assess the impact of antibiotics and vaccines, on H. influenzae carriage rates, carriage density, and disease.
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                Author and article information

                Contributors
                Journal
                Front Public Health
                Front Public Health
                Front. Public Health
                Frontiers in Public Health
                Frontiers Media S.A.
                2296-2565
                27 March 2020
                2020
                : 8
                : 95
                Affiliations
                Department of Clinical Microbiology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine , Shanghai, China
                Author notes

                Edited by: Kai Zhou, Zhejiang University, China

                Reviewed by: Maria Giufre, Higher Institute of Health (ISS), Italy; Yuetian Yu, Shanghai JiaoTong University, China; Fredrik Resman, Lund University, Sweden; Seyed Davar Siadat, Pasteur Institute of Iran (PII), Iran

                *Correspondence: Li-Zhong Han hanlizhong1107@ 123456163.com

                This article was submitted to Infectious Diseases - Surveillance, Prevention and Treatment, a section of the journal Frontiers in Public Health

                †These authors have contributed equally to this work and share first authorship

                Article
                10.3389/fpubh.2020.00095
                7135888
                32292774
                5886aaf9-c293-451e-a6fa-94a2f37cc114
                Copyright © 2020 Li, Xiao, Gu, He, Ni and Han.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 03 September 2019
                : 06 March 2020
                Page count
                Figures: 2, Tables: 5, Equations: 0, References: 35, Pages: 8, Words: 4974
                Categories
                Public Health
                Original Research

                haemophilus influenzae,molecular epidemiology,drug resistance,multilocus sequence type,ftsi gene

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