Resistant Haemophilus Influenzae Research Articles

First global report about the prevalence of multi-drug resistant Haemophilus influenzae: a systematic review and meta-analysis

BackgroundIn recent decades, the prevalence of antibiotic resistance is increasing in Haemophilus influenzae (Haemophilus influenzae), which poses important challenges to global health. This research offers a comprehensive meta-analysis of the global epidemiology of multi-drug resistant (MDR) H. influenzae.MethodsIn this study, we conducted a meta-analysis based on PRISMA checklist. Electronic databases including PubMed, ISI Web of Science, Scopus, EMBASE, and Google Scholar were reviewed using keywords related to H. influenzae and antibiotic resistance. Eligible studies were selected based on stringent inclusion and exclusion criteria. Then, data from these studies were analyzed using the Comprehensive Meta-Analysis (CMA) software.ResultsOf 375 retrieved articles, 16 met the inclusion criteria. These studies were conducted from 2003 to 2023 and analyzed data from 19,787 clinical isolates of H. influenzae. The results showed different levels of resistance of H. influenzae to different antibiotics: ampicillin (36%), azithromycin (15.3%), ceftriaxone (1.4%), etc. The global prevalence for beta-lactamases producing H. influenzae and MDR H. influenzae was measured 34.9% and 23.1%, respectively. The prevalence rate of MDR H. influenzae was higher in Asian countries (24.6%) compared to Western regions (15.7%). MDR H. influenzae had the highest prevalence in meningitis cases (46.9%) and the lowest prevalence in acute otitis media (0.5%).ConclusionsThe prevalence of MDR H. influenzae has been increasing worldwide, especially in Asian regions. This highlights the urgent need for monitoring and implementation of effective antibiotic stewardship programs globally.

Characterization and Fitness Cost of Tn7100, a Novel Integrative and Conjugative Element Conferring Multidrug Resistance in Haemophilus influenzae.

A multidrug-resistant (MDR) strain of Haemophilus influenzae, Hi-228, with phenotypic resistance toward ampicillin, cefotaxime, chloramphenicol, gentamicin, and azithromycin, was isolated in Oslo, Norway. The strain was part of a clonal outbreak (2016–2017) comprising five ST143 strains with identical resistotypes. Hi-228 carries a novel integrative and conjugative element (ICE), Tn7100, contributing to this remarkable and previously unreported MDR profile. Tn7100 contains the following resistance genes: blaTEM−1B, catA2, aac(6′)-Im, aph(2″)-Ib, mef (E), and mel. The latter four are previously unreported or rarely reported in H. influenzae. In this study, we investigated the genetic environment, mechanisms of transfer, impact on phenotypic susceptibility, and fitness cost of this ICE. We found that Tn7100 has an overall structure similar to the previously described ICE Tn6686, with blaTEM−1B and catA2 carried by Tn3 and Tn10, respectively. The major difference between Tn7100 and Tn6686 is that Tn7100 lacks tet(B) but carries the resistance gene pairs aac(6′)-Im and aph(2″)-Ib and mef (E) and mel. The gene pairs are located on the novel transposable elements Tn7470 and Tn7471, which have high sequence identities to a plasmid in Enterobacterales and an ICE in streptococcal species, respectively. Tn7100 does circularize and is transferable, however, at a low frequency. Head-to-head competition experiments showed that uptake of Tn7100 reduces bacterial fitness. Our study shows that MDR strains are capable of clonal spread and that the H. influenzae supragenome comprises an increasingly wide range of transferable resistance genes, with evidence of transfer from unrelated genera. The findings offer a glimpse into the genome dynamics of H. influenzae, highlighting the importance of rational antibiotic usage to contain antimicrobial resistance and the emergence of MDR strains in this important pathogen.

Evaluation of the in vitro activity of ceftaroline, ceftazidime/avibactam and comparator antimicrobial agents against clinical isolates from paediatric patients in Kuwait: ATLAS data 2012-19.

To report antimicrobial resistance data for Gram-positive and Gram-negative pathogens isolated from paediatric patients in three hospitals in Kuwait during 2012-19. In vitro activity of antimicrobials against isolates from documented infections was determined using CLSI broth microdilution method and breakpoints at a central laboratory. Enterobacterales and Pseudomonas aeruginosa isolates were screened for β-lactamases using multiplex PCR assays. Phenotypic determination of resistance in Haemophilus influenzae and Gram-positive isolates was performed using standard methodologies. Among 515 Enterobacterales isolates, 29.3% were ESBL-positive; susceptibility was highest to amikacin, ceftazidime/avibactam and meropenem (≥97.4%), regardless of ESBL status. CTX-M-15 was identified in 87.1% of ESBL-positive Escherichia coli and 84.2% of ESBL-positive Klebsiella pneumoniae isolates. Of 111 P. aeruginosa isolates, 9.9% were MDR and 12.6% meropenem-resistant (MEM-R). Amikacin and ceftazidime/avibactam had the highest susceptibility rates in the overall group (≥92.8%), with reduced rates among MDR and MEM-R isolates. All 269 MRSA and 180 MSSA isolates were susceptible to daptomycin, linezolid, teicoplanin, tigecycline and vancomycin. All MSSA and 99.3% of MRSA were ceftaroline susceptible. All 168 pneumococcal isolates were susceptible to ceftaroline, linezolid, tigecycline and vancomycin. H. influenzae and Streptococcus pyogenes ceftaroline susceptibility rates were ≥93.3% and ≥95.6%. Most isolates of Enterobacterales (including resistant phenotypes) and P. aeruginosa from Kuwait during 2012-19 were susceptible to ceftazidime/avibactam. Ceftaroline was active against most Gram-positive isolates, including resistant phenotypes, and ESBL-negative Enterobacterales. These results indicate that novel antibiotics such as ceftazidime/avibactam and ceftaroline represent valuable treatment options for paediatric infections, including those caused by MDR organisms.

Evaluation of the Haemophilus influenzae EUCAST and CLSI disc diffusion methods to recognize aminopenicillin and amoxicillin/clavulanate resistance.

Implementation of EUCAST susceptibility testing in an Australian hospital laboratory demonstrated higher rates of aminopenicillin and amoxicillin/clavulanate resistance in Haemophilus influenzae than previously recognized. This study aimed to better define the variability in the detection of β-lactam resistance based on EUCAST and CLSI disc diffusion (DD) methodology, by comparison with the recommended reference method, broth microdilution (BMD), and by concordance with genomic analysis. A total of 100 random H. influenzae isolates were assessed for ampicillin and amoxicillin/clavulanate susceptibility by EUCAST and CLSI DD and BMD. WGS was used to analyse the ftsI gene of a subset of isolates with β-lactam resistance, other than that due to isolated β-lactamase production. Of the 100 isolates, 32 were categorized as either β-lactamase negative, ampicillin resistant (BLNAR) (n = 18) or β-lactamase positive, amoxicillin/clavulanate resistant (BLPACR) (n = 14) by EUCAST DD. All 18 EUCAST BLNAR isolates were genotypically confirmed by WGS. Five of 18 BLNAR isolates were concordant by CLSI DD, 12 by EUCAST BMD and 4 by CLSI BMD. Nine of 14 EUCAST BLPACR isolates were confirmed by WGS; the remaining 5 were 1 mm below the EUCAST DD breakpoint. Only one isolate was detected as BLPACR by CLSI DD. Group III mutations associated with high-level ampicillin resistance were identified in 10/32 isolates. The EUCAST DD susceptibility method is more reliable than either CLSI or BMD for the detection of genotypically defined BLNAR resistance. However, accurate categorization of amoxicillin/clavulanate resistance remains problematic. Continuous and reproducible surveillance of resistance is needed; for this to be possible, robust susceptibility methods are required.

Molecular epidemiology of imipenem resistance in invasive Haemophilus influenzae infections in Germany in 2016

The carbapenems imipenem and meropenem play an important role in the empirical anti-infective treatment of critically ill patients. Carbapenem resistance in Haemophilus influenzae (Hi) has rarely been reported. We provide prevalence data for resistance to carbapenems from laboratory surveillance of invasive Hi infections in Germany in 2016. Phenotypic susceptibility testing against ampicillin, amoxicillin/clavulanate, cefotaxime and imipenem was carried out on 474 isolates from blood and CSF. The isolates were collected as part of the national laboratory surveillance programme. Imipenem-resistant strains were further tested for meropenem susceptibility. Molecular analysis was done by ftsI sequencing to detect mutations in PBP3, by acrR sequencing to detect alterations in the regulatory protein of the AcrAB-TolC efflux pump and by MLST. No resistance to meropenem was detected. Cefotaxime resistance was rare (n = 3; 0.6%). Imipenem resistance was found in 64 strains (13.5%) using gradient agar diffusion and was confirmed in 26 isolates by broth microdilution (5.5%). Imipenem resistance occurred predominantly in Hi that were β-lactamase negative but ampicillin resistant and in those that were β-lactamase positive but nevertheless amoxicillin/clavulanate resistant. This finding suggested a β-lactamase-independent mechanism. Accordingly, sequence analysis of PBP3 identified previously described mutations. MLST of the imipenem-resistant strains, which were all non-typeable Hi, revealed a high diversity. We conclude that imipenem, but not meropenem, resistance is frequent in Hi. It is likely to be supported by PBP3 mutations.

Fluoroquinolone resistance in Haemophilus influenzae from nursing home residents in Taiwan: correlation of MICs and mutations in QRDRs.

To study the association between number and positions of mutations with MICs of fluoroquinolone non-susceptible Haemophilus influenzae. More than 40% of 48 H. influenzae isolated from nursing home residents were not susceptible to fluoroquinolone. Amino acid changes in the quinolone resistance determining regions, and correlation with MICs and inhibition zone diameters were analysed. All isolates with reduced susceptibility to fluoroquinolones (MIC ≥0·125µgml-1 ) had at least one mutation in gyrA at position 84 and were resistant to nalidixic acid. Compared to isolates with reduced susceptibility, resistant isolates were associated with mutations in gyrA at positions 88 and 134, and in parC at position 88 (P<0·001). Inhibition zone diameter for nalidixic acid disk ≥23mm may detect susceptible isolates. Reduced susceptibility to fluoroquinolones was associated with mutations at position 84 in gyrA. A further increase in fluoroquinolone MIC was associated with mutations in gyrA at positions 88 and 134, and parC at position 88. Due to limited resistant H. influenzae strains, prior studies on association between positions of mutations and fluoroquinolone MICs were inconclusive. The comparison of mutations between isolates with susceptibility, reduced susceptibility and high resistance supported the importance of the present study.

Molecular characterization of fluoroquinolones, macrolides, and imipenem resistance in Haemophilus influenzae: analysis of the mutations in QRDRs and assessment of the extent of the AcrAB-TolC-mediated resistance.

The aims of the present study were to characterize the mechanisms of resistance to fluoroquinolones, macrolides, and imipenem in Haemophilus influenzae, to assess the extent of the AcrAB-TolC-mediated resistance, and to define a core genome multilocus sequence typing (cgMLST) scheme for H. influenzae by using whole-genome sequencing. Four amino acid substitutions in GyrA (at Ser84 and Asp88), ParC (at Ser84), and ParE (at Asp420) were found to be closely associated to the MICs. We did not find any amino acid substitution surrounding the three highly conserved amino acid motifs in PBP3 related to imipenem resistance. All the isolates possessed the ermB gene. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) decreased the MIC of imipenem by twofold for FQR-6 and fourfold for GE47 and GE88 strains. For erythromycin, the MICs were decreased by twofold. We found that the six FQR isolates were clustered in two groups. The number of different loci within FQR-1_FQR-3_FQR-5 cluster was 6, while FQR-2 and FQR-4 differed for 21 loci. FQR-1_FQR-3_FQR-5 and FQR-2_FQR-4 clusters were distant among each other and compared to 19 genomes downloaded from NCBI, to 8 strains heteroresistant to imipenem, and to 4 strains monoresistant to ciprofloxacin isolated in Denmark. We confirmed that specific amino acid substitutions in GyrA, ParC, and ParE are implicated in quinolone resistance. Additionally, the degree of resistance is related to the number of these amino acid substitutions. We provide robust evidence that drug efflux is one of the substantial mechanisms of imipenem and erythromycin resistance in H. influenzae.

Resistance decay in individuals after antibiotic exposure in primary care: a systematic review and meta-analysis

BackgroundAntibiotic resistance is an urgent global problem, but reversibility is poorly understood. We examined the development and decay of bacterial resistance in community patients after antibiotic use.MethodsThis was a systematic review and meta-analysis. PubMed, EMBASE and CENTRAL (from inception to May 2017) were searched, with forward and backward citation searches of the identified studies. We contacted authors whose data were unclear, and of abstract-only reports, for further information. We considered controlled or times-series studies of patients in the community who were given antibiotics and where the subsequent prevalence of resistant bacteria was measured. Two authors extracted risk of bias and data. The meta-analysis used a fixed-effects model.ResultsOf 24,492 articles screened, five controlled and 20 time-series studies (total 16,353 children and 1461 adults) were eligible.Resistance in Streptococcus pneumoniae initially increased fourfold after penicillin-class antibiotic exposure [odds ratio (OR) 4.2, 95% confidence interval (CI) 3.5–5.4], but this fell after 1 month (OR 1.7, 95% CI 1.3–2.1). After cephalosporin-class antibiotics, resistance increased (OR 2.2, 95%CI 1.7-2.9); and fell to (OR 1.6, 95% CI 1.2-2.3) at 1 month. After macrolide-class antibiotics, resistance increased (OR 3.8, 95% CI 1.9–7.6) and persisted for 1 month (OR 5.2, 95% CI 2.6–10.3) and 3 months (OR 8.1, 95% CI 4.6–14.2, from controlled studies and OR 2.3, 95% CI 0.6–9.4, from time-series studies).Resistance in Haemophilus influenzae after penicillins was not significantly increased (OR 1.3, 95% CI 0.9–1.9) initially but was at 1 month (OR 3.4, 95% CI 1.5–7.6), falling after 3 months (OR 1.0, 95% CI 0.5–2.2). Data were sparse for cephalosporins and macrolides.Resistance in Enterobacter increased post-exposure (OR 3.2, 95% CI 0.9–10.8, from controlled studies and OR 7.1, 95% CI 4.2–12, from time-series studies], but was lower after 1 month (OR 1.8, 95% CI 0.9–3.6).ConclusionsResistance generally increased soon after antibiotic use. For some antibiotic classes and bacteria, it partially diminished after 1 and 3 months, but longer-term data are lacking and urgently needed.Trial registrationPROSPERO CRD42015025499.

Levofloxacin and moxifloxacin resistant Haemophilus influenzae in a patient with common variable immunodeficiency (CVID): a case report

Fluoroquinolones are important in the treatment of respiratory infections and resistance to them amongst Haemophilus influenzae is rare. We report a case of persistent respiratory tract colonization with an H. influenzae strain resistant to ciprofloxacin, levofloxacin and moxifloxacin. Two sputum cultures obtained 4.5 months apart, from a patient with common variable immunodeficiency (CVID), yielded H. influenzae in colony counts exceeding 107 CFU/ml. Antibiotic susceptibility testing using disk diffusion and E-tests revealed both isolates to be resistant to ciprofloxacin, levofloxacin and moxifloxacin. Although quinolone resistant H. influenzae remains extremely rare, its emergence in Sri Lanka heralds microbiological and clinical challenges with important implications for the treatment of community acquired respiratory infection.

Children with respiratory tract infections in Swedish primary care; prevalence of antibiotic resistance in common respiratory tract pathogens and relation to antibiotic consumption

BackgroundThe majority of antibiotics consumed in developed countries are prescribed in primary care. However, little is known about resistance levels in the primary care population.MethodNasopharyngeal cultures were obtained from children, 0-10 years of age, seeking care at their Primary Health Care Centre with symptoms of respiratory tract infection. Parental questionnaires were used to retrieve information about the child’s previous antibiotic consumption.ResultCultures from 340 children were gathered. The level of resistant Haemophilus influenzae was low and the prevalence of penicillin non-susceptible pneumococci (PNSP MIC ≥ 0.125 mg/L) was 6% compared to 10% (p = 0.31) in corresponding cultures from children diagnosed at the local clinical microbiology laboratory. Antibiotic treatment within the previous 4 weeks predisposed for resistant bacteria in the nasopharynx, OR: 3.08, CI 95% (1.13-8.42).ConclusionLow prevalence of PNSP supports the use of phenoxymethylpenicillin as empirical treatment for childhood upper respiratory tract infections attending primary care in our setting. It is important that studies on resistance are performed in primary care populations to evaluate data from microbiological laboratories. Recent antibiotic treatment increases risk of bacterial resistance in children and continuous work to reduce unnecessary antibiotic prescribing should be prioritised.

Effect of Fluoroquinolones and Macrolides on Eradication and Resistance of Haemophilus influenzae in Chronic Obstructive Pulmonary Disease.

Little is known about the effect of antibiotics on eradication of carriage and development of resistance in Haemophilus influenzae in individuals with chronic obstructive pulmonary disease (COPD). Our goals were to assess antibiotic susceptibilities, prevalence of resistance genes, and development of resistance in H. influenzae and to evaluate the effect of macrolide and fluoroquinolone administration on H. influenzae eradication. Data were from a 15-year longitudinal study of COPD. Genome sequence data were used to determine genotype and identify resistance genes. MICs of antibiotics were determined by reference broth microdilution. Generalized linear mixed models were used to evaluate associations between antibiotic use and H. influenzae eradication. We examined 267 H. influenzae isolates from 77 individuals. All newly acquired H. influenzae isolates were susceptible to azithromycin. Five of 27 (19%) strains developed 4-fold increases in azithromycin MICs and reached or exceeded the susceptibility breakpoint (≤4 μg/ml) during exposure. H. influenzae isolates were uniformly susceptible to ciprofloxacin, levofloxacin, and moxifloxacin (MIC90s of 0.015, 0.015, and 0.06, respectively); there were no mutations in quinolone resistance-determining regions. Fluoroquinolone administration was associated with increased H. influenzae eradication compared to macrolides (odds ratio [OR], 16.67; 95% confidence interval [CI], 2.67 to 104.09). There was no difference in H. influenzae eradication when comparing macrolide administration to no antibiotic (OR, 1.89; 95% CI, 0.43 to 8.30). Fluoroquinolones are effective in eradicating H. influenzae in individuals with COPD. Macrolides are ineffective in eradicating H. influenzae, and their use in COPD patients may lead to decreased macrolide susceptibility and resistance.

The infections and resistance of Haemophilus influenzae

Haemophilus influenzae (Hi) is an important human pathogen that causes severe infections including meningitis, bacteremic pneumonia and sepsis, mostly affecting young children. The resistance of Hi to antimicro-bials has risen and wide variations exist in antibiotic resistance, both geographically and over time. The main mechanisms of resistance of Hi are those to β-lactam antibiotics. Antibiotic utilization is the main risk factors for the deve-lopment of antimicrobial resistance. Third-generation cephalosporins is the most effective antibiotic agent to treat the infections of Hi in children population. With continued research, progress toward a broadly effective vaccine to prevent infections caused by Hi is expected in the near future. Key words: Haemophilus influenzae; Antibiotic resistance; Child

Emergence of clonally related multidrug resistant Haemophilus influenzae with penicillin-binding protein 3-mediated resistance to extended-spectrum cephalosporins, Norway, 2006 to 2013.

Resistance to cephalosporins in Haemophilus influenzae is usually caused by characteristic alterations in penicillin-binding protein 3 (PBP3), encoded by the ftsI gene. Resistance to extended-spectrum cephalosporins is associated with high-level PBP3-mediated resistance (high-rPBP3), defined by the second stage S385T substitution in addition to a first stage substitution (R517H or N526K). The third stage L389F substitution is present in some high-rPBP3 strains. High-rPBP3 H. influenzae are considered rare outside Japan and Korea. In this study, 30 high-rPBP3 isolates from Norway, collected between 2006 and 2013, were examined by serotyping, multilocus sequence typing (MLST), ftsI sequencing, detection of beta-lactamase genes and minimum inhibitory concentration (MIC) determination. MICs were interpreted according to clinical breakpoints from the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Respiratory isolates predominated (proportion: 24/30). The 30 isolates included one serotype f isolate, while the remaining 29 lacked polysaccharide capsule genes. Resistance to extended-spectrum cephalosporins (cefixime, 29 isolates/30 isolates; cefepime, 28/30; cefotaxime, 26 /30; ceftaroline, 26/30; ceftriaxone, 14/30), beta-lactamase production (11/30) and co-resistance to non-beta-lactams (trimethoprim-sulfamethoxazole, 13/30; tetracycline, 4/30; chloramphenicol, 4/30; ciprofloxacin, 3/30) was frequent. The N526K substitution in PBP3 was present in 23 of 30 isolates; these included a blood isolate which represents the first invasive S385T + N526K isolate reported from Europe. The L389F substitution, present in 16 of 30 isolates, coincided with higher beta-lactam MICs. Non-susceptibility to meropenem was frequent in S385T + L389F + N526K isolates (8/12). All 11 beta-lactamase positive isolates were TEM-1. Five clonal groups of two to 10 isolates with identical MLST-ftsI allelic profiles were observed, including the first reported high-rPBP3 clone with TEM-1 beta-lactamase and co-resistance to ciprofloxacin, tetracycline, chloramphenicol and trimethoprim-sulfamethoxazole. Prior to this study, no multidrug resistant high-rPBP3 H. influenzae had been reported in Norway. Intensified surveillance of antimicrobial resistance is needed to guide empiric therapy.

Levofloxacin-resistant haemophilus influenzae, Taiwan, 2004-2010.

Levofloxacin resistance in Haemophilus influenzae has increased significantly in Taiwan, from 2.0% in 2004 to 24.3% in 2010 (p<0.001). Clinical and molecular investigations of 182 levofloxacin-resistant isolates revealed that the increase was mainly the result of the spread of several clones in the elderly population in different regions.

Role of inter-species recombination of the ftsI gene in the dissemination of altered penicillin-binding-protein-3-mediated resistance in Haemophilus influenzae and Haemophilus haemolyticus

To screen the ftsI gene sequences obtained from clinical isolates of non-typeable Haemophilus influenzae (NTHi) and Haemophilus haemolyticus for the presence of mosaic ftsI gene structures, and to evaluate the role of inter-species recombination of the ftsI gene in the formation and distribution of resistant ftsI genes. The ftsI genes of 100 Haemophilus isolates comprising genetically defined β-lactamase-negative ampicillin-susceptible (gBLNAS), β-lactamase-positive ampicillin-resistant (gBLPAR), β-lactamase-negative ampicillin-resistant (gBLNAR) and β-lactamase-positive amoxicillin/clavulanate-resistant (gBLPACR) isolates of NTHi (n = 50) and H. haemolyticus (n = 50) were analysed in this study. Both the flanking regions and the full-length ftsI gene sequences of all study isolates were screened for mosaic structures using H. influenzae Rd and H. haemolyticus ATCC 33390 as reference parental sequences, and bioinformatics methods were used for recombination analysis using SimPlot. Of the 100 clinical isolates analysed 34% (34/100) harboured mosaic ftsI gene structures containing distinct ftsI gene fragments similar to both reference parental sequences. The inter-species recombination events were exclusively encountered in the ftsI gene of gBLNAR/gBLPACR isolates of both NTHi and H. haemolyticus, and were always associated with the formation of a mosaic fragment at the 3' end of the ftsI gene. There was no evidence supporting horizontal gene transfer (HGT) involving the entire ftsI gene among the clinical isolates in vivo. We provide evidence for the HGT and inter-species recombination of the ftsI gene among gBLNAR/gBLPACR isolates of NTHi and H. haemolyticus in a clinical setting, highlighting the importance of recombination of the ftsI gene in the emergence of altered penicillin-binding protein 3 and BLNAR-mediated resistance.

A molecular analysis of quinolone-resistant Haemophilus influenzae: Validation of the mutations in Quinolone Resistance-Determining Regions

The mechanism of quinolone-resistance is considered to be amino acid mutations in the type II topoisomerase. We validated the genetic mechanisms of quinolone resistance in Haemophilus influenzae. We obtained 29 H.influenzae strains from a nationwide surveillance program in Japan (including 11 quinolone-resistant strains [moxifloxacin: MFLX or levofloxacin MIC ≥2μg/ml]). We analyzed the sequences of the Quinolone Resistance-Determining Regions (QRDRs) in GyrA, GyrB, ParC and ParE. Furthermore, we induced resistance in susceptible strains by exposing them to quinolone, and investigated the relationship between mutations in the QRDRs and the MICs. Five amino acid substitutions in GyrA (at Ser84 and Asp88) and ParC (at Gly82, Ser84 and Glu88) were found to be closely related to the MICs. The strains with a MFLX MIC of 0.125-1 and 2-4μg/ml had one and two mutations, respectively. The strains with a MFLX MIC of ≥8μg/ml had three or more mutations. The strains with induced resistance with MFLX MICs of 0.5-1 and ≥2μg/ml also had one and two mutations, respectively. We confirmed that these five mutations strongly contribute to quinolone resistance and found that the degree of resistance is related to the number of the mutations. In addition, the three strains of 18 susceptible strains (16.7%) also had a single mutation. These strains may therefore be in the initial stage of quinolone resistance. Currently, the frequency of quinolone-resistant H.influenzae is still low. However, as has occurred with β-lactams, an increase in quinolone use may lead to more quinolone-resistant strains.

Multilocus sequence typing and ftsI sequencing: a powerful tool for surveillance of penicillin-binding protein 3-mediated beta-lactam resistance in nontypeable Haemophilus influenzae.

BackgroundBeta-lactam resistance in Haemophilus influenzae due to ftsI mutations causing altered penicillin-binding protein 3 (PBP3) is increasing worldwide. Low-level resistant isolates with the N526K substitution (group II low-rPBP3) predominate in most geographical regions, while high-level resistant isolates with the additional S385T substitution (group III high-rPBP3) are common in Japan and South Korea.Knowledge about the molecular epidemiology of rPBP3 strains is limited. We combined multilocus sequence typing (MLST) and ftsI/PBP3 typing to study the emergence and spread of rPBP3 in nontypeable H. influenzae (NTHi) in Norway.ResultsThe prevalence of rPBP3 in a population of 795 eye, ear and respiratory isolates (99% NTHi) from 2007 was 15%. The prevalence of clinical PBP3-mediated resistance to ampicillin was 9%, compared to 2.5% three years earlier. Group II low-rPBP3 predominated (96%), with significant proportions of isolates non-susceptible to cefotaxime (6%) and meropenem (20%). Group III high-rPBP3 was identified for the first time in Northern Europe.Four MLST sequence types (ST) with characteristic, highly diverging ftsI alleles accounted for 61% of the rPBP3 isolates. The most prevalent substitution pattern (PBP3 type A) was present in 41% of rPBP3 isolates, mainly carried by ST367 and ST14. Several unrelated STs possessed identical copies of the ftsI allele encoding PBP3 type A.Infection sites, age groups, hospitalization rates and rPBP3 frequencies differed between STs and phylogenetic groups.ConclusionsThis study is the first to link ftsI alleles to STs in H. influenzae. The results indicate that horizontal gene transfer contributes to the emergence of rPBP3 by phylogeny restricted transformation.Clonally related virulent rPBP3 strains are widely disseminated and high-level resistant isolates emerge in new geographical regions, threatening current empiric antibiotic treatment. The need of continuous monitoring of beta-lactam susceptibility and a global system for molecular surveillance of rPBP3 strains is underlined. Combining MLST and ftsI/PBP3 typing is a powerful tool for this purpose.

成人急性鼻副鼻腔炎における薬剤耐性菌検出の背景因子に関する検討

In the treatment of adult patients with acute rhinosinusitis, it has been assumed that subjects over 65 years of age or living with children attending a day nursery, are at higher risk for infection with drug-resistant bacteria. However there are few reports which have discussed the relationship between those risk factors and adult patients with acute rhinosinusitis. I investigated the association between adult patients with acute rhinosinusitis living with children attending a day nursery and gender, ages, and frequency of detection of drug-resistant bacteria. From 2010 to 2012, a total of 598 adult patients with acute rhinosinusitis were enrolled. The majority of the study subjects were women, and the largest 10-year age group was 30-39 years. By sex, a greater proportion of the female subjects than the male subjects lived with children attending a day nursery. By age, the proportion of subjects who lived with children attending a day nursery was significantly higher in the 30-39-year age group than other age groups. Drug-resistant Streptococcus pneumoniae and ampicillin (ABPC)-resistant Haemophilus influenzae were detected at significantly higher rates in subjects with children attending a day nursery than in those without. There were no significant difference in drug-resistant bacteria detection between subjects over 65 years of age, and under 65 years. These findings suggested that living with children attending a day nursery is a risk factor and source of infection by drug-resistant organisms that may cause intra-familial infections of adult patients with acute rhinosinusitis. These results suggested we must ask adult patients with acute rhinosinusitis whether they are living with children attending a day nursery or not, when we plan their treatment strategy.

High prevalence of nasal carriage of β-lactamase-negative ampicillin-resistantHaemophilus influenzaein healthy children in Korea

This study investigated the carriage of antimicrobial resistant Haemophilus influenzae in 582 healthy children attending kindergarten or elementary school at four intervals over a 9-month period in Seoul, Korea. Diverse colonization patterns and a lower level of long-term persistent carriage by H. influenzae status were evident in this study. Colonizing H. influenzae isolates showed a high rate of resistance to β-lactams including ampicillin (51·9%), cefaclor (52·1%), and amoxicillin/clavulanate (16·3%). Based on the ampicillin resistance mechanism, H. influenzae isolates were categorized as β-lactamase-negative, ampicillin-susceptible (BLNAS) (48·1%), β-lactamase-positive, ampicillin-resistant (BLPAR) (22·6%), β-lactamase-negative, ampicillin-resistant (BLNAR) (22·8%), and β-lactamase-positive, amoxicillin/clavulanate-resistant (BLPACR) strains (6·5%). This study provides the first evidence of a high prevalence (22·8%) of BLNAR strains of H. influenzae nasal carriage in healthy children attending kindergarten or the first 2 years of elementary school in Korea. The high carriage of these resistant strains in overcrowded urban settings may create reservoirs for development of H. influenzae-resistant strains.

Contribution of ROB-1 and PBP3 mutations to the resistance phenotype of a β-lactamase-positive amoxicillin/clavulanic acid-resistant Haemophilus influenzae carrying plasmid pB1000 in Italy

plasmid pB1000 bearing bla(ROB-1) is responsible for high-level β-lactam resistance in Haemophilus influenzae as well as in Pasteurella multocida and Haemophilus parasuis isolates from Spain. Here, we explore the presence of ROB-1 in Italy and investigate the relative contribution of penicillin-binding protein 3 (PBP3) mutations and ROB-1 to the β-lactam resistance phenotype in H. influenzae. the collection of the Italian Reference Laboratory of H. influenzae was investigated for ROB-1-positive isolates between 2004 and 2009. H. influenzae Rd KW20 was used as recipient for pB1000 electroporation and for mutagenesis of the ftsI gene encoding PBP3. the presence of plasmid pB1000 in a non-typeable H. influenzae isolated in Italy, BB1059, is reported in this work. This strain is not genetically related to the H. influenzae clinical isolates bearing pB1000 described in Spain. The sequence of ftsI from BB1059 revealed several mutations in the predicted amino acid sequence of PBP3. To determine the relative contribution of pB1000 and PBP3 mutations to the β-lactam resistance phenotype of BB1059, H. influenzae Rd KW20 was transformed with ftsI and/or pB1000 from BB1059. β-Lactam resistance profiles revealed the additive effect of pB1000 and PBP3 mutations conferring resistance to β-lactams, including amoxicillin/clavulanic acid and third-generation cephalosporins. intra-European spread of plasmid pB1000 among H. influenzae has been shown. The coexistence of plasmid pB1000 and mutations in PBP3 produces an additive resistance phenotype in H. influenzae.