Molecular characteristics and biofilm formation capacity of nontypeable Haemophilus influenza strains isolated from lower respiratory tract in children.

With the widespread introduction of the Hib conjugate vaccine, Nontypeable Haemophilus influenzae (NTHi) has emerged as the predominant strain globally. NTHi presents a significant challenge as a causative agent of chronic clinical infections due to its high rates of drug resistance and biofilm formation. While current research on NTHi biofilms in children has primarily focused on upper respiratory diseases, investigations into lower respiratory sources remain limited. In this study, we collected 54 clinical strains of lower respiratory tract origin from children. Molecular information and drug resistance features were obtained through whole gene sequencing and the disk diffusion method, respectively. Additionally, an in vitro biofilm model was established. All clinical strains were identified as NTHi and demonstrated the ability to form biofilms in vitro. Based on scanning electron microscopy and crystal violet staining, the strains were categorized into weak and strong biofilm-forming groups. We explored the correlation between biofilm formation ability and drug resistance patterns, as well as clinical characteristics. Stronger biofilm formation was associated with a longer cough duration and a higher proportion of abnormal lung imaging findings. Frequent intake of ß-lactam antibiotics might be associated with strong biofilm formation. While a complementary relationship between biofilm-forming capacity and drug resistance may exist, further comprehensive studies are warranted. This study confirms the in vitro biofilm formation of clinical NTHi strains and establishes correlations with clinical characteristics, offering valuable insights for combating NTHi infections.

Whole-genome analysis of Haemophilus influenzae strains isolated from persons with cystic fibrosis.

Introduction. Haemophilus influenzae is a commensal of the respiratory tract that is frequently present in cystic fibrosis (CF) patients and may cause infection. Antibiotic resistance is well described for CF strains, and virulence factors have been proposed.Hypothesis/Gap. The genetic diversity of H. influenzae strains present in the lungs of persons with CF is largely unknown despite the fact that this organism is considered to be a pathogen in this condition. The aim was to establish the genetic diversity and susceptibility of H. influenzae strains from persons with CF, and to screen the whole genomes of these strains for the presence of antibiotic resistance determinants and proposed virulence factors.Methods. A total of 67 strains, recovered from respiratory samples from persons with CF from the UK (n=1), Poland (n=2), Spain (n=24) and the Netherlands (n=40), were subjected to whole-genome sequencing using Illumina technology and tested for antibiotic susceptibility. Forty-nine of these strains (one per different sequence type) were analysed for encoded virulence factors and resistance determinants.Results. The 67 strains represented 49 different sequence types. Susceptibility testing showed that all strains were susceptible to aztreonam, ciprofloxacin, imipenem and tetracycline. Susceptibility to ampicillin, ampicillin/sulbactam, amoxicillin/clavulanic acid, cefuroxime, cefixime, ceftriaxone, cefepime, meropenem, clarithromycin, co-trimoxazole and levofloxacin ranged from 70.2-98.5%. Only 6/49 strains (12.2%) harboured acquired resistance genes. Mutations associated with a ß-lactamase-negative ampicillin-resistant phenotype were present in four strains (8.2 %). The potential virulence factors, urease, haemoglobin- and haptoglobin-binding protein/carbamate kinase, and OmpP5 (OmpA), were encoded in more than half of the strains. The genes for HMW1, HMW2, H. influenzae adhesin, a IgA-specific serine endopeptidase autotransporter precursor, a TonB-dependent siderophore, an ABC-transporter ATP-binding protein, a methyltransferase, a BolA-family transcriptional regulator, glycosyltransferase Lic2B, a helix-turn-helix protein, an aspartate semialdehyde dehydrogenase and another glycosyltransferase were present in less than half of the strains.Conclusion. The H. influenzae strains showed limited levels of resistance, with the highest being against co-trimoxazole. Sequences encoding a carbamate kinase and a haemoglobin- and haemoglobin-haptoglobin-binding-like protein, a glycosyl transferase and an urease may aid the colonization of the CF lung. The adhesins and other identified putative virulence factors did not seem to be necessary for colonization.

Comparative pangenome analysis of capsulated Haemophilus influenzae serotype f highlights their high genomic stability.

Haemophilus influenzae is an opportunistic pathogen adapted to the human respiratory tract. Non-typeable H. influenzae are highly heterogeneous, but few studies have analysed the genomic variability of capsulated strains. This study aims to examine the genetic diversity of 37 serotype f isolates from the Netherlands, Portugal, and Spain, and to compare all capsulated genomes available on public databases. Serotype f isolates belonged to CC124 and shared few single nucleotide polymorphisms (SNPs) (n = 10,999), but a high core genome (> 80%). Three main clades were identified by the presence of 75, 60 and 41 exclusive genes for each clade, respectively. Multi-locus sequence type analysis of all capsulated genomes revealed a reduced number of clonal complexes associated with each serotype. Pangenome analysis showed a large pool of genes (n = 6360), many of which were accessory genome (n = 5323). Phylogenetic analysis revealed that serotypes a, b, and f had greater diversity. The total number of SNPs in serotype f was significantly lower than in serotypes a, b, and e (p < 0.0001), indicating low variability within the serotype f clonal complexes. Capsulated H. influenzae are genetically homogeneous, with few lineages in each serotype. Serotype f has high genetic stability regardless of time and country of isolation.

Haemophilus influenzae Serotype a as a Cause of Meningitis in Children in Brazil.

BACKGROUND: Since the introduction of Haemophilus influenzae type b vaccines, invasive disease due to Haemophilus influenzae serotype a (Hia) has been reported with increasing frequency. METHODS: This study is based on hospital-based surveillance for Hia meningitis over a 5-year period. RESULTS: Thirty-five patients with H. influenzae meningitis were hospitalized and 12 were serotype a. Hia was detected in blood and cerebrospinal fluid by culture or reverse transcription polymerase chain reaction. Patients' median age was 10 months, 7 (58%) boys and 5 (41%) girls. Ten (83%) children had received at least 1 vaccine dose against Haemophilus influenzae type b. All patients were treated with ceftriaxone for a median period of 11 days. The main complications described were empyema in 5 (41%) and seizures in 3 (25%) patients. Two (16.6%) patients died due to cerebral damage and shock. CONCLUSIONS: Invasive disease due to Hia affecting young children accounts for considerable morbidity and mortality.

Epidemiology and population structure of Haemophilus influenzae causing invasive disease.

This study provides an update on invasive Haemophilus influenzae disease in Bellvitge University Hospital (2014-2019), reporting its evolution from a previous period (2008-2013) and analysing the non-typeable H. influenzae (NTHi) population structure using a clade-related classification. Clinical data, antimicrobial susceptibility and serotyping were studied and compared with those of the previous period. Population structure was assessed by multilocus sequence typing (MLST), SNP-based phylogenetic analysis and clade-related classification. The incidence of invasive H. influenzae disease remained constant between the two periods (average 2.07 cases per 100 000 population), while the 30 day mortality rate decreased (20.7-14.7 %, respectively). Immunosuppressive therapy (40 %) and malignancy (36 %) were the most frequent comorbidities. Ampicillin and fluoroquinolone resistance rates had increased between the two periods (10-17.6 % and 0-4.4 %, respectively). NTHi was the main cause of invasive disease in both periods (84.3 and 85.3 %), followed by serotype f (12.9 and 8.8 %). NTHi displayed high genetic diversity. However, two clusters of 13 (n=20) and 5 sequence types (STs) (n=10) associated with clade V included NTHi strains of the most prevalent STs (ST3 and ST103), many of which showed increased frequency over time. Moreover, ST103 and ST160 from clade V were associated with ß-lactam resistance. Invasive H. influenzae disease is uncommon, but can be severe, especially in the elderly with comorbidities. NTHi remains the main cause of invasive disease, with ST103 and ST160 (clade V) responsible for increasing ß-lactam resistance over time.

Increased Incidence of Invasive Haemophilus influenzae Disease Driven by Non-Type B Isolates in Ontario, Canada, 2014 to 2018.

Haemophilus influenzae can cause serious invasive disease. We report the epidemiology and antimicrobial susceptibility of invasive H. influenzae in Ontario, Canada, from 2014 to 2018 from laboratory-based data. Blood was the most common specimen source (89.5%). Consistent with widespread vaccination against serotype b (Hib), the incidence of Hib in Ontario remained low (0.04 cases per 100,000 population). H. influenzae disease primarily afflicted those <1 and ≥65 years of age. From 2014 to 2018, cases of invasive H. influenzae increased 5.6%, from 1.67 to 2.06 cases per 100,000 population, the majority of which were attributed to a 7.6% increase in the incidence of H. influenzae in those ≥65 years old. H. influenzae disease was primarily caused by nontypeable H. influenzae (NTHi) (74.2%) and, to a much lesser extent, serotype a (Hia) (8.9%) and serotype f (Hif) (10.2%). Serotype-dependent trends in antimicrobial susceptibility were observed. Hia and Hif isolates were predominantly susceptible to all antibiotics tested, while 27.2% of NTHi isolates were nonsusceptible to ampicillin. Resistance to ceftriaxone and meropenem, first-line antibiotics for invasive disease treatment, was nonexistent. The incidence of invasive H. influenzae in Ontario is increasing. The incidence and antimicrobial susceptibility of all serotypes and nontypeable H. influenzae should be monitored. IMPORTANCE H. influenzae can cause serious invasive, life-threatening disease and is considered 1 of 12 priority pathogens by the World Health Organization. Widespread vaccination against H. influenzae serotype b (Hib) has resulted in very low incidence of Hib in Ontario and other regions that have vaccination programs. However, the epidemiology of non-Hib serotypes and nontypeable H. influenzae (NTHi) remains poorly understood. Here, we describe the epidemiology of all invasive H. influenzae isolates (N = 1,338) received by our laboratory over the 5-year period and report on the antimicrobial susceptibility patterns by serotype. Overall, we observed an increase in the incidence of invasive disease over the study period, primarily driven by NTHi. Serotype-dependent trends in antimicrobial susceptibility were also observed. This work contributes to the global understanding of H. influenzae epidemiology and antimicrobial resistance and is additionally important for further vaccine planning initiatives.

A LAMP-based system for rapid detection of eight common pathogens causing lower respiratory tract infections.

Lower respiratory tract infections (LRTIs) are a leading cause of morbidity and mortality worldwide and lack a rapid diagnostic method. To improve the diagnosis of LRTIs, we established an available loop-mediated isothermal amplification (LAMP) assay for the detection of eight common lower respiratory pathogens, including Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli, Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. The whole process can be achieved within 1 h (sample to results read out). We established an extraction free isothermal system. 528 sputum samples collected from patients suspected to have LRTIs were analyzed by the system (8 tests in each sample, a total of 4224 tests) and compared with the standard culture method (SCM). The samples with inconsistent results were further verified by Sanger sequencing and High-throughput sequencing (NGS). The detection limits of the LAMP assay for the 8 pathogens ranged from 103 to 104 CFU/mL. Upon testing 528 samples, the Kappa coefficients of all pathogens ranged between 0.5 and 0.7 indicated a moderate agreement between the LAMP assay and the SCM. All inconsistent samples were further verified by Sanger sequencing, we found that the developed LAMP assay had a higher consistency level with Sanger sequencing than the SCM for all pathogens. Additionally, when the NGS was set to a diagnostic gold standard, the specificity and sensitivity of the LAMP assay for LRTIs were 94.49% and 75.00%. The present study demonstrated that the developed LAMP has high consistency with the sequencing methods. Meanwhile, the LAMP assay has a higher detection rate compared to the SCM. It may be a powerful tool for rapid and reliable clinical diagnosis of LRTIs in primary hospitals.

Molecular Epidemiology of Contemporary Invasive Haemophilus influenzae Isolates in Texas Children.

Although vaccination has reduced the incidence of Haemophilus influenzae type b, nontypeable H. influenzae and other encapsulated types remain a health threat. Little is known regarding the contemporary molecular epidemiology of these organisms. We conducted multilocus sequence typing on invasive H. influenzae during a period of increasing incidence.

Haemophilus influenzae Prevalence, Proportion of Capsulated Strains and Antibiotic Susceptibility During Colonization and Acute Otitis Media in Children, 2019-2020.

BACKGROUND: The objective of this study was to determine the prevalence, proportion of encapsulated strains and antibiotic susceptibility of Haemophilus influenzae isolated from young children. METHODS: Children, 6 months to 30 months old, were prospectively enrolled from September 2019 to September 2020 at Rochester, NY, pediatric clinics. H. influenzae isolates from nasopharynx (NP) at healthy visits and disease isolates from NP and middle ear fluid (MEF) at onset of acute otitis media (AOM) were characterized by capsular typing, ß-lactamase production and antibiotic susceptibility. RESULTS: Samples from 565 healthy visits and 130 AOM visits were collected. H. influenzae was detected 5.9% and 27% in the NP from healthy and AOM visits, respectively. In the MEF, H. influenzae was isolated in 43% of samples. Eight percent of H. influenzae isolates were encapsulated, 88% type f. Overall 39.7% of isolates were ß-lactamase producing; 43% for MEF isolates. Ampicillin, trimethoprim/sulfamethoxazole, erythromycin and clarithromycin nonsusceptibility were found in more than 25% of isolates. None of the encapsulated H. influenzae isolates were positive for ß-lactamase production or ampicillin nonsusceptibility. 9.2% of isolates were ß-lactamase negative, ampicillin resistant (ß-lactamase negative, ampicillin resistant + ß-lactamase negative, ampicillin intermediate). CONCLUSIONS: The prevalence of H. influenzae in the NP of young children is very low at times of health, but H. influenzae is highly prevalent in MEF at onset of AOM. Nontypeable H. influenzae accounts for >90% of all H. influenzae isolates. Type f predominated among encapsulated strains. ß-lactamase production and antibiotic nonsusceptibility among H. influenzae strains isolated from the NP and MEF are common.

Haemophilus influenzae one day in Denmark: prevalence, circulating clones, and dismal resistance to aminopenicillins.

Haemophilus influenzae is a common cause of mucosal infections that warrants accurate surveillance. We aimed to assess the prevalence of the species in clinical specimens, and characterise population structure and resistance to aminopenicillins by whole genome sequencing.We assessed the point prevalence by entering the database records of 1 day in Denmark and examined the genome sequences of nationwide, collected isolates from the same day. The prevalence of H. influenzae in clinical samples on the 10th of January 2018 was 1.78 per 100,000 person-days (all samples), and 2.47 per 1000 hospital bed-days (hospital samples). Of 2009 bacteria deemed clinically relevant and collected in a concerted action by the Danish departments of clinical microbiology, 62 (3.1%) were H. influenzae. All 62 isolates belonged to phylogenetic group I and were unencapsulated. Three strains from separate Danish regions had identical core genome sequences, but a small number of intergenic mutations testified to circulating clones, rather than individual cases of patient-to-patient transmission. The TEM-1 ß-lactamase gene was present in 24 strains, while 13 strains were genetically categorised as ampicillin-resistant due to substitutions in penicillin-binding protein 3; shared patterns of amino acid substitutions in unrelated strains indicated putative lateral transfer of chromosomal resistance. Circulating clones of H. influenzae are frequent, and host factors, rather than direct transmission of epidemic strains, may be the primary cause of infection. The bleak presence of ampicillin resistance revealed by sequencing of point prevalence strains underscores the necessity for close examination of testing methods.

Analysis of Haemophilus species in patients with respiratory tract infections in Yaoundé, Cameroon.

OBJECTIVES: To identifyHaemophilus species and characterize antimicrobial susceptibility of isolates from patients with respiratory tract infections (RTIs) in Cameroon. METHODS: Isolates (n = 95) were from patients with RTIs obtained from two Hospitals in Yaoundé, Cameroon. Isolates were identified by biochemical assay, PCR-based method, MALDI-TOF and whole genome sequencing. Antibiotic minimum inhibitory concentrations were determined by E-test. RESULTS: H. influenzae was the most prevalent species varying from 76.8% to 84.2% according to different methods. The isolates were mainly nontypable (n = 70, 96%). Three isolates of H. influenzae were capsulated (b, e and f). The isolates were genetically diverse and 40 unique sequence types were identified including 11 new ones. Resistance to ampicillin was observed among 55.3% (52/94) and 9% (14/52) produced TEM-1 ß-lactamase. PBP3 mutations occurred in 57.7% of ampicillin resistant isolates (30/52). Eleven isolates were chloramphenicol resistant with 80% producing chloramphenicol acetyltransferase (8/10). Four Haemophilus isolates were rifampicin resistant with two mutations in rpoB gene. Five isolates were ciprofloxacin resistant and harbored mutations in the quinolone resistance determining regions of gyrA and parC genes. CONCLUSION: H. influenzae isolates are highly diverse and show high levels of antibiotic resistance. H. influenzae serotype b is still circulating in the post-vaccination era.

Invasive non-typeable Haemophilus influenzae infection due to endometritis associated with adenomyosis.

BACKGROUND: The widespread administration of the Haemophilus influenzae type b vaccine has led to the predominance of non-typable H. influenzae (NTHi). However, the occurrence of invasive NTHi infection based on gynecologic diseases is still rare. CASE PRESENTATION: A 51-year-old Japanese woman with a history of adenomyoma presented with fever. Blood cultures and a vaginal discharge culture were positive with NTHi. With the high uptake in the uterus with 67Ga scintigraphy, she was diagnosed with invasive NTHi infection. In addition to antibiotic administrations, a total hysterectomy was performed. The pathological analysis found microabscess formations in adenomyosis. CONCLUSIONS: Although NTHi bacteremia consequent to a microabscess in adenomyosis is rare, this case emphasizes the need to consider the uterus as a potential source of infection in patients with underlying gynecological diseases, including an invasive NTHi infection with no known primary focus.

Nontypeable Haemophilus influenzae Responds to Virus-Infected Cells with a Significant Increase in Type IV Pilus Expression.

Nontypeable Haemophilus influenzae (NTHI) colonizes the human nasopharynx, but when the host immune response is dysregulated by upper respiratory tract (URT) virus infection, NTHI can gain access to more distal airway sites and cause disease. The NTHI type IV pilus (T4P) facilitates adherence, benign colonization, and infection, and its majority subunit PilA is in clinical trials as a vaccinogen. To further validate the strategy of immunization with PilA against multiple NTHI-induced diseases, it is important to demonstrate T4P expression under microenvironmental conditions that predispose to NTHI infection of the airway. Because URT infection commonly facilitates NTHI-induced diseases, we examined the influence of ongoing virus infection of respiratory tract epithelial cells on NTHI T4P expression in vitro Polarized primary human airway epithelial cells (HAEs) were sequentially inoculated with one of three common URT viruses, followed by NTHI. Use of a reporter construct revealed that NTHI upregulated pilA promoter activity when cultured with HAEs infected with adenovirus (AV), respiratory syncytial virus (RSV), or rhinovirus (RV) versus that in mock-infected HAEs. Consistent with these results, pilA expression and relative PilA/pilin abundance, as assessed by quantitative reverse transcription-PCR (qRT-PCR) and immunoblot, respectively, were also significantly increased when NTHI was cultured with virus-infected HAEs. Collectively, our data strongly suggest that under conditions of URT virus infection, PilA vaccinogen induction of T4P-directed antibodies is likely to be highly effective against multiple NTHI-induced diseases by interfering with T4P-mediated adherence. We hypothesize that this outcome could thereby limit or prevent the increased load of NTHI in the nasopharynx that characteristically precedes these coinfections.IMPORTANCE Nontypeable Haemophilus influenzae (NTHI) is the predominant bacterial causative agent of many chronic and recurrent diseases of the upper and lower respiratory tracts. NTHI-induced chronic rhinosinusitis, otitis media, and exacerbations of cystic fibrosis and chronic obstructive pulmonary disease often develop during or just after an upper respiratory tract viral infection. We have developed a vaccine candidate immunogen for NTHI-induced diseases that targets the majority subunit (PilA) of the type IV twitching pilus (T4P), which NTHI uses to adhere to respiratory tract epithelial cells and that also plays a role in disease. Here, we showed that NTHI cocultured with virus-infected respiratory tract epithelial cells express significantly more of the vaccine-targeted T4P than NTHI that encounters mock-infected (healthy) cells. These results strongly suggest that a vaccine strategy that targets the NTHI T4P will be effective under the most common predisposing condition: when the human host has a respiratory tract virus infection.

Extensively drug-resistant Haemophilus influenzae - emergence, epidemiology, risk factors, and regimen.

BACKGROUND: Concern about Haemophilus influenzae infection has been increasing over recent decades. Given the emergence of H. influenzae with severe drug resistance, we assessed the prevalence of as well as risk factors and potential therapies for extensively drug-resistant (XDR) H. influenzae infection in Taiwan. RESULTS: In total, 2091 H. influenzae isolates with disk diffusion-based antibiotic susceptibility testing from 2007 to 2018 were enrolled. H. influenzae strains resistant to ampicillin, chloramphenicol, levofloxacin, and trimethoprim-sulfamethoxazole tended to be isolated from patient wards (≧41%), whereas those resistant to amoxicillin-clavulanate, cefotaxime, and cefuroxime were more likely to be isolated from intensive care units (approximately 50%). XDR H. influenzae was first identified in 2007, and its incidence did not significantly change thereafter. Overall prevalence of single, multiple, and extensively drug-resistant H. influenzae over 2007-2018 was 21.5% (n = 450), 26.6% (n = 557), and 2.5% (n = 52), respectively. A stepwise logistic regression analysis revealed that blood culture (odds ratio: 4.069, 95% confidence intervals: 1.339-12.365, P = 0.013) was an independent risk factor for XDR H. influenzae infection. No nosocomial transmission of XDR H. influenzae observed. Antibiotic susceptibility testing results demonstrated that cefotaxime was effective against 78.8% (n = 41) of the XDR strains. CONCLUSIONS: The presence of XDR H. influenzae strains was identified in Taiwan, and cefotaxime was efficacious against most of these strains.

Two cases of type-a Haemophilus influenzae meningitis within the same week in the same hospital are phylogenetically unrelated but recently exchanged capsule genes.

Haemophilus influenzae causes common and sometimes severe adult and pediatric disease including chronic obstructive respiratory disease, otitis media and infections of the central nervous system. Serotype b strains, with a b-type capsule, have been the historical cause of invasive disease, and the introduction of a serotype b-specific vaccine has led to their decline. However, unencapsulated or non-b-type H. influenzae infections are not prevented by the vaccine and appear to be increasing in frequency. Here we report two pediatric cases of severe central nervous system H. influenzae infection presenting to the same hospital in San Diego, California during the same week in January 2016. Due to good vaccine coverage in this part of the world, H. influenzae cases are normally rare and seeing two cases in the same week was unexpected. We thus suspected a recent transmission chain, and possible local outbreak. To test this hypothesis, we isolated and sequenced whole genomes from each patient and placed them in a phylogenetic tree spanning the known diversity of H. influenzae. Surprisingly, we found that the two isolates (SD2016_1 and SD2016_2) belonged to distantly related lineages, suggesting two independent transmission events and ruling out a local outbreak. Despite being distantly related, the two isolates belong to two different lineages that have exchanged capsule loci in the recent past. Therefore, as in other bacterial pathogens, capsule switching by horizontal gene transfer may be an important evolutionary mechanism of vaccine evasion in H. influenzae.

Co-carriage of Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis among three different age categories of children in Hungary.

BACKGROUND: The nasopharynx can from time to time accommodate otherwise pathogenic bacteria. This phenomenon is called asymptomatic carriage. However, in case of decreased immunity, viral infection or any other enhancing factors, severe disease can develop. Our aim in this study was to survey the nasal carriage rates of four important respiratory pathogens in three different age groups of children attending nurseries, day-care centres and primary schools. This is the first study from Hungary about the asymptomatic carriage of H. influenzae and M. catarrhalis. METHODS: Altogether 580 asymptomatic children were screened in three Hungarian cities. Samples were collected from both nostrils with cotton swabs. The identification was based on both colony morphology and species-specific PCRs. Serotyping was performed for S. pneumoniae, H. influenzae and M. catarrhalis. Antibiotic susceptibility was determined with agar dilution, according to the EUCAST guidelines. Clonality was examined by PFGE. RESULTS AND CONCLUSIONS: Whereas the carriage rates of S. pneumoniae, H. influenzae and M. catarrhalis clearly decreased with age, that of S. aureus showed an opposite tendency. Multiple carriage was least prevalent if S. aureus was one of the participants. The negative association between this bacterium and the others was statistically significant. For pneumococcus, the overall carriage rate was lower compared to earlier years, and PCV13 serotypes were present in only 6.2% of the children. The majority of H. influenzae isolates was non-typeable and no type b was detected; serotype A was dominant among M. catarrhalis. All four bacteria were more sensitive to antibiotics compared to clinical isolates. No MRSAs were detected, but we found three mupirocin resistant strains. The positive effect of Hib- and PCV-vaccination is undoubted. Continued surveillance of these pathogens is required.

Characterization of biofilm formation and induction of apoptotic DNA fragmentation by nontypeable Haemophilus influenzae on polarized human airway epithelial cells.

Nontypeable Haemophilus influenzae (NTHi) is a common airway commensal and opportunistic pathogen that persists within biofilm communities in vivo. Biofilm studies so far are mainly based on assays on plastic surfaces. The aim of this work was to investigate the capacity of clinical NTHi strains to form biofilm structures on polarized Calu-3 human airway epithelial cells and primary normal human bronchial epithelial cells and to characterize the biofilm architecture. Formation of adherent NTHi biofilms post colonization of host cells at multiple time-points was evaluated using confocal laser scanning microscopy and electron microscopy. NTHi biofilms were analyzed in terms of biofilm height and presence of extracellular matrix components, and their apoptotic effects on epithelial cells were measured by TUNEL assay. Strain Fi176 was observed to form robust biofilms on airway epithelia over time, while disrupting the integrity of Calu-3 monolayer by 72 h of co-culture. NTHi biofilms were observed to induce apoptotic DNA fragmentation in host cells at 24 h post infection. Biofilm formation on cell monolayers by Fi176ΔpilA strain was markedly reduced compared to WT strain. Biofilm inhibition and disruption assays by crystal violet staining indicated that DNA and proteins are part of NTHi biofilms in vitro. Our findings highlight critical stages of NTHi pathogenesis following host colonization and provide useful biofilm models for future antimicrobial drug discovery investigations.

Molecular epidemiology and evolution of Haemophilus influenzae.

Haemophilus influenzae remains a common cause of illness in children worldwide. H. influenzae type b is the leading cause of bacterial meningitis in children before introduction of vaccination and is a common cause of pneumonia, epiglottis and septic arthritis. Since the implementation of the Hib conjugate vaccine, the non-typeable H. influenzae has rapidly decreased in respiratory and invasive infections in children and adults. However, the rate of antibiotic resistance of H. influenzae varies with region and period and is usually on the rise. In this review, typing of H. influenzae, virulence factors and resistance will be dissertated.

First outbreak of Haemophilus influenzae clone ST422 with low susceptibility to quinolones in paediatric patients in Japan.

Introduction. Recently, a Haemophilus influenzae clone with low susceptibility to quinolones emerged in paediatric patients in Japan. Isolates of this clone survived for a long time when exposed to the therapeutic concentration of quinolones, despite being classified as 'susceptible' under the criteria of the Clinical and Laboratory Standards Institute. In the present study, we report the first outbreak of this clone in paediatric patients in 2018.Aim. Our aim was to characterise the first outbreak of an H. influenzae clone with low susceptibility to quinolones.Methodology. All H. influenzae isolates (n=62), collected at a Japanese teaching hospital in 2018, were characterized by both antimicrobial susceptibility tests and multilocus sequence typing. In addition, the similarity in genetic backgrounds was analysed by PFGE.Results. Among all the isolates (n=62), quinolone low-susceptible isolates accounted for 19.4 % (n=12). Seven out of 12 isolates were identified as sequence type 422 (ST422) and showed more than 90 % similarity to each other by PFGE analysis. All ST422 isolates exhibited identical amino acid substitutions in both quinolone resistance-determining regions in GyrA and ParC. In addition, all these isolates were from paediatric patients who had been referred by different primary care clinics and had no relationship to each other.Conclusion. In this study, we describe an outbreak of a quinolone low-susceptible ST422 clone in paediatric patients in Japan. Because ST422 isolates have already been reported in at least five other countries, it has the potential to spread worldwide.