A multi-iron enzyme installs copper-binding oxazolone/thioamide pairs on a nontypeable Haemophilus influenzae virulence factor.

The multinuclear nonheme iron-dependent oxidases (MNIOs) are a rapidly growing family of enzymes involved in the biosynthesis of ribosomally synthesized, posttranslationally modified peptide natural products (RiPPs). Recently, a secreted virulence factor from nontypeable Haemophilus influenzae (NTHi) was found to be expressed from an operon, which we designate the hvf operon, that also encodes an MNIO. Here, we show by Mössbauer spectroscopy that the MNIO HvfB contains a triiron cofactor. We demonstrate that HvfB works together with HvfC [a RiPP recognition element (RRE)-containing partner protein] to perform six posttranslational modifications of cysteine residues on the virulence factor precursor peptide HvfA. Structural characterization by tandem mass spectrometry and NMR shows that these six cysteine residues are converted to oxazolone and thioamide pairs, similar to those found in the RiPP methanobactin. Like methanobactin, the mature virulence factor, which we name oxazolin, uses these modified residues to coordinate Cu(I) ions. Considering the necessity of oxazolin for host cell invasion by NTHi, these findings point to a key role for copper during NTHi infection. Furthermore, oxazolin and its biosynthetic pathway represent a potential therapeutic target for NTHi.

Evaluation of a multiplex-qPCR for paediatric pleural empyema-An observational study in hospitalised children.

Pleural empyema is a serious complication of pneumonia in children. Negative bacterial cultures commonly impede optimal antibiotic therapy. To improve bacterial identification, we developed a molecular assay and evaluated its performance compared with bacterial culture. Our multiplex-quantitative PCR to detect Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus and Haemophilus influenzae was assessed using bacterial genomic DNA and laboratory-prepared samples (n = 267). To evaluate clinical performance, we conducted the Molecular Assessment of Thoracic Empyema (MATE) observational study, enrolling children hospitalised with empyema. Pleural fluids were tested by bacterial culture and multiplex-qPCR, and performance determined using a study gold standard. We determined clinical sensitivity and time-to-organism-identification to assess the potential of the multiplex-qPCR to reduce the duration of empiric untargeted antibiotic therapy. Using spiked samples, the multiplex-qPCR demonstrated 213/215 (99.1%) sensitivity and 52/52 (100%) specificity for all organisms. During May 2019-March 2023, 100 children were enrolled in the MATE study; median age was 3.9 years (IQR 2-5.6). A bacterial pathogen was identified in 90/100 (90%) specimens by multiplex-qPCR, and 24/100 (24%) by bacterial culture (P <0.001). Multiplex-qPCR identified a bacterial cause in 68/76 (90%) culture-negative specimens. S. pneumoniae was the most common pathogen, identified in 67/100 (67%) specimens. We estimate our multiplex-qPCR would have reduced the duration of untargeted antibiotic therapy in 61% of cases by a median 20 days (IQR 17.5-23, range 1-55). Multiplex-qPCR significantly increased pathogen detection compared with culture and may allow for reducing the duration of untargeted antibiotic therapy.

Membranome-based identification of amino acid substitution in Haemophilus influenzae multidrug efflux pump HmrM for reduced chloramphenicol susceptibility.

A decreased chloramphenicol susceptibility in Haemophilus influenzae is commonly caused by the activity of chloramphenicol acetyltransferases (CATs). However, the involvement of membrane proteins in chloramphenicol susceptibility in H. influenzae remains unclear. In this study, chloramphenicol susceptibility testing, whole-genome sequencing, and analyses of membrane-related genes were performed in 51 H. influenzae isolates. Functional complementation assays and structure-based protein analyses were conducted to assess the effect of proteins with sequence substitutions on the minimum inhibitory concentration (MIC) of chloramphenicol in CAT-negative H. influenzae isolates. Six isolates were resistant to chloramphenicol and positive for type A-2 CATs. Of these isolates, A3256 had a similar level of CAT activity but a higher chloramphenicol MIC relative to the other resistant isolates; it also had 163 specific variations in 58 membrane genes. Regarding the CAT-negative isolates, logistic regression and receiver operator characteristic curve analyses revealed that 48T > G (Asn16Lys), 85 C > T (Leu29Phe), and 88 C > A (Leu30Ile) in HI_0898 (emrA), and 86T > G (Phe29Cys) and 141T > A (Ser47Arg) in HI_1177 (artM) were associated with enhanced chloramphenicol susceptibility, whereas 997G > A (Val333Ile) in HI_1612 (hmrM) was associated with reduced chloramphenicol susceptibility. Furthermore, the chloramphenicol MIC was lower in the CAT-negative isolates with EmrA-Leu29Phe/Leu30Ile or ArtM-Ser47Arg substitution and higher in those with HmrM-Val333Ile substitution, relative to their counterparts. The Val333Ile substitution was associated with enhanced HmrM protein stability and flexibility and increased chloramphenicol MICs in CAT-negative H. influenzae isolates. In conclusion, the substitution in H. influenzae multidrug efflux pump HmrM associated with reduced chloramphenicol susceptibility was characterised.

Increase in invasive Haemophilus influenzae serotype A infections during the COVID-19 pandemic in New South Wales, Australia.

Haemophilus influenzae, a causative agent of severe invasive infections such as meningitis, sepsis and pneumonia, is classified into encapsulated or typeable (represented by serotypes A to F) and non-typeable varieties (NTHi) by the presence or absence of the polysaccharide capsule. Invasive disease caused by H. influenzae type B (HIB) can be prevented through vaccination which remains the main disease control intervention in many countries. This study examined the genomic diversity of circulating H. influenzae strains associated with invasive disease in New South Wales, Australia, before and during the COVID-19 pandemic. Ninety-six isolates representing 95 cases of invasive H. influenzae infections (iHi) diagnosed between January 2017 and September 2022 were typed and characterised using whole genome sequencing. These cases were caused by serotypes A (n=24), B (n=35), E (n=3), F (n=2) and NTHi (n=32). There was an apparent decline in the number of iHi infections during the COVID-19 pandemic, with a corresponding increase in the proportion of iHi cases caused by serotype A (HIA), which returned to pre-pandemic levels in 2022. Fifteen isolates associated with HIB or non-typeable iHi were resistant to ß-lactams due to a PBP3 mutation or carriage of blaTEM-1. Further, capsular gene duplication was observed in HIB isolates but was not found in HIA. These findings provide important baseline genomic data for ongoing iHi surveillance and control.

A novel biosensing strategy for identification of three important bacteria causing meningitis.

Bacterial meningitis is an acute infection which requires rapid diagnosis and treatment due to the high mortality and serious consequences of the disease. The purpose of this study was to design a homemade multiplex PCR and a novel fluorescence biosensor on chip (FBC) to detect three important agents of meningitis including Streptococcus pneumoniae (S. pneumoniae), Neisseria meningitidis (N. meningitidis), and Haemophilus influenzae (H. influenzae). The homemade multiplex PCR can diagnose three bacterial species simultaneously. Fabrication of FBC was carried out based on the deposition of lead nanoparticles on a quartz slide using the thermal evaporation method. Then, the SH-Cap Probe/Target ssDNA /FAM-Rep probe was loaded on lead film. The evaluation of the fluorescence reaction when the probes bind to the target ssDNA was assessed by a Cytation 5 Cell Imaging Multimode Reader Bio-Tek. The limit of detections (LOD) in homemade PCR and FBC to identify S. pneumoniae were 119 × 102 CFU/mL (0.27 ng/µL) and 380 CFU/mL (9 pg/µL), respectively. The LODs of homemade PCR and FBC for detection of N. meningitidis were 4.49 CFU/mL (1.1 pg/µL) and 13 × 103 CFU/mL (30 pg/µL), respectively. Our results confirmed the LODs of homemade PCR and FBC in detection of H. influenzae were 15.1 CFU/mL (30 fg/µL) and 41 × 102 CFU/mL (90 pg/ µL), respectively. Both techniques had appropriate sensitivity and specificity in detection of S. pneumoniae, N. meningitidis and H. influenzae.

Exploring the pediatric nasopharyngeal bacterial microbiota with culture-based MALDI-TOF mass spectrometry and targeted metagenomic sequencing.

The nasopharynx is an important reservoir of disease-associated and antimicrobial-resistant bacterial species. This proof-of-concept study assessed the utility of a combined culture, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), and targeted metagenomic sequencing workflow for the study of the pediatric nasopharyngeal bacterial microbiota. Nasopharyngeal swabs and clinical metadata were collected from Cambodian children during a hospital outpatient visit and then biweekly for 12 weeks. Swabs were cultured on chocolate and blood-gentamicin agar, and all colony morphotypes were identified by MALDI-TOF MS. Metagenomic sequencing was done on a scrape of all colonies from a chocolate agar culture and processed using the mSWEEP pipeline. One hundred one children were enrolled, yielding 620 swabs. MALDI-TOF MS identified 106 bacterial species/40 genera: 20 species accounted for 88.5% (2,190/2,474) of isolates. Colonization by Moraxella catarrhalis (92.1% of children on ≥1 swab), Haemophilus influenzae (87.1%), and Streptococcus pneumoniae (83.2%) was particularly common. In S. pneumoniae-colonized children, a median of two serotypes [inter-quartile range (IQR) 1-2, range 1-4] was detected. For the 21 bacterial species included in the mSWEEP database and identifiable by MALDI-TOF, detection by culture + MALDI-TOF MS and culture + mSWEEP was highly concordant with a median species-level agreement of 96.9% (IQR 86.8%-98.8%). mSWEEP revealed highly dynamic lineage-level colonization patterns for S. pneumoniae which were quite different to those for S. aureus. A combined culture, MALDI-TOF MS, targeted metagenomic sequencing approach for the exploration of the young child nasopharyngeal microbiome was technically feasible, and each component yielded complementary data. IMPORTANCE: The human upper respiratory tract is an important source of disease-causing and antibiotic-resistant bacteria. However, understanding the interactions and stability of these bacterial populations is technically challenging. We used a combination of approaches to determine colonization patterns over a 3-month period in 101 Cambodian children. The combined approach was feasible to implement, and each component gave complementary data to enable a better understanding of the complex patterns of bacterial colonization.

Microbial aetiology of community-acquired pneumonia in hospitalised adults: A prospective study utilising comprehensive molecular testing.

OBJECTIVES: This study aimed to describe the microbial aetiology of community-acquired pneumonia (CAP) in adults admitted to a tertiary care hospital and assess the impact of syndromic polymerase chain reaction (PCR) panels on pathogen detection. METHODS: Conducted at Haukeland University Hospital, Norway, from September 2020 to April 2023, this prospective study enrolled adults with suspected CAP. We analysed lower respiratory tract samples using both standard-of-care tests and the BIOFIRE® FILMARRAY® Pneumonia Plus Panel (FAP plus). The added value of FAP Plus in enhancing the detection of clinically relevant pathogens, alongside standard-of-care diagnostics, was assessed. RESULTS: Of the 3238 patients screened, 640 met the inclusion criteria, with 384 confirmed to have CAP at discharge. In these patients, pathogens with proven or probable clinical significance were identified in 312 (81.3%) patients. Haemophilus influenzae was the most prevalent pathogen, found in 118 patients (30.7%), followed by SARS-CoV-2 in 74 (19.3%), and Streptococcus pneumoniae in 64 (16.7%). Respiratory viruses were detected in 186 (48.4%) patients. The use of FAP plus improved the pathogen detection rate from 62.8% with standard-of-care methods to 81.3%. CONCLUSIONS: Pathogens were identified in 81% of CAP patients, with Haemophilus influenzae and respiratory viruses being the most frequently detected pathogens. The addition of the FAP plus panel, markedly improved pathogen detection rates compared to standard-of-care diagnostics alone.

Characterization of Ceftriaxone-Resistant Haemophilus influenzae Among Korean Children.

BACKGROUND: Haemophilus influenzae is a frequently encountered pathogen responsible for respiratory tract infections in children. Following the detection of ceftriaxone-resistant H. influenzae at our institution, we aimed to investigate the resistance mechanisms of ceftriaxone in H. influenzae, with a particular focus on alterations in penicillin-binding protein 3 (PBP3) and ß-lactamase production. METHODS: Among H. influenzae isolates collected at Asan Medical Center Children's Hospital from March 2014 to April 2019, ceftriaxone-resistant strains by the disk-diffusion test were included. Ceftriaxone minimum inhibitory concentrations (MICs) were determined using the E-test according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The presence of ß-lactamase was assessed through cefinase test and TEM-1/ROB-1 polymerase chain reaction (PCR). PBP3 alterations were explored via ftsI gene sequencing. RESULTS: Out of the 68 collected strains, 21 exhibited resistance to ceftriaxone in disk diffusion tests. Two strains were excluded due to failed subculture. Among 19 ceftriaxone-resistant H. influenzae isolates, eighteen were non-typeable H. influenzae, and twelve were positive for TEM-1 PCR. Isolates were classified into groups II (harboring only N526K, n = 3), III (N526K+S385T, n = 2), III+ (S385T+L389F+N526K, n = 11), and III-like+ (S385T+L389F+R517H, n = 3) according to the PBP3 alteration pattern. With a median ceftriaxone MIC of 0.190 mg/L (range, 0.008-0.750), the median ceftriaxone MIC was the highest in group III-like+ (0.250 mg/L), followed by groups III+ (0.190 mg/L), III (0.158 mg/L), and II (0.012 mg/L). All three strains belonging to group II, which did not harbor the S385T substitution, had ceftriaxone MICs of ≤ 0.125 mg/L. CONCLUSION: The emergence of ceftriaxone-resistant H. influenzae with ceftriaxone MIC values of up to 0.75 mg/L was observed even in children in South Korea, with most associated with S385T and L389F substitutions. The N526K mutation alone does not significantly impact ceftriaxone resistance. Further large-scale studies are essential to investigate changes in antibiotic resistance patterns and factors influencing antibiotic resistance in H. influenzae isolated from pediatric patients in Korea.

Ampicillin susceptibility testing of Haemophilus influenzae in the routine clinical laboratory by the EUCAST methodology compared to broth microdilution and the presence of ftsI gene mutations.

OBJECTIVES: To investigate the prevalence of ampicillin resistance in Haemophilus influenzae and the diagnostic accuracy of the EUCAST recommended disc diffusion method to detect the increasingly prevalent ampicillin resistance due to the presence of PBP3 alterations based on mutations in the ftsI gene. METHODS: During a 6-month period all consecutive non-duplicate H. influenzae isolates were prospectively collected and stored. MICs of ampicillin were determined by broth microdilution (BMD). PCR was performed to detect mutations in the ftsI gene. Results of routine disc diffusion susceptibility testing, including the penicillin screening test in accordance with the current EUCAST methodology, as well as additional Etest results, were compared to the BMD as the reference method. RESULTS: In 102 isolates, the prevalence of ampicillin resistance was 28% (29/102) by BMD. There was a good correlation between MICs of ampicillin and the presence of a ß-lactamase and/or an ftsI gene mutation. The prevalence of ampicillin resistance was overestimated using the EUCAST method (33% (34/102)) and underestimated when an additional Etest was used (24% (24/102)) (not significant). The sensitivity and specificity of the EUCAST methodology for the detection of ampicillin resistance were 97% ((28/29); 95% CI, 82-100%) and 92% ((67/73); 95% CI, 83-97%), respectively. CONCLUSIONS: The prevalence of ampicillin resistance was 28%, as determined by BMD. Although the overall diagnostic accuracy of the EUCAST ampicillin disc diffusion was high, misclassification of ampicillin susceptibility may still occur.

Recurrent community-acquired pneumococcal meningitis in adults with and without identified predisposing factors.

Bacterial meningitis is still a significant public health concern, with high morbidity and mortality rates. Despite this, it is still a rare event that requires the bacterial invasion of the meninges. However, some predisposing factors can trigger recurrent episodes of meningitis. This study is aimed at determining the clinical characteristics and the molecular epidemiology of episodes of recurrent community-acquired meningitis with and without predisposing factors. For this purpose, we performed a retrospective study of our laboratory database during the period of 2010 to 2020. Additionally, using molecular tools developed in our previous works, the epidemiology of the pathogens causing these episodes was analyzed using cerebrospinal fluid samples, especially in the absence of isolated strains. We observed a total of 1,779 meningitis cases and 230 were caused by Streptococcus pneumoniae. Of those, 16 were recurrent meningitis episodes (16/1,779; 0.9%) from seven patients. Pneumococcus was the main agent responsible in these recurrent episodes and only two episodes were caused by Haemophilus influenzae. The mean age of these patients was 20 years old and three had predisposing factors which could have led to contracting meningitis. The samples presented different pneumococcal serotypes. Most of them were non-vaccine-covered serotypes and antibiotic susceptible strains. Therefore, it was demonstrated how the practical employment of molecular tools, developed for research, when applied in the routine of diagnosis, can provide important information for epidemiological surveillance. Furthermore, it was shown how pneumococcus was the leading cause of recurrent community-acquired meningitis without predisposing factors, suggesting that pneumococcal vaccination may be necessary, even in those groups of individuals considered to be less susceptible.

Preparation and immunogenicity evaluation of C-HapS-P6 fusion protein vaccine against nontypeable Haemophilus influenzae in mice.

Nontypeable Haemophilus influenzae (NTHi) is the dominant pathogen in several infectious diseases. Currently the use of antibiotics is the main intervention to prevent NTHi infections, however with the emergence of drug resistant strains, it has compromised the treatment of respiratory infections with antibiotics. Therefore there is an urgent need to develop a safe and effective vaccine to prevent NTHi infections. We investigate the potential of C-HapS-P6 fusion protein as a vaccine for treating NTHi in murine models. PGEX-6P2/C-HapS-P6 fusion gene was constructed using overlap extension polymerase chain reaction. The recombined plasmid was transformed into Escherichia coli for protein expression. The mice were subjected to intraperitoneal immunization using purified antigens. Immunoglobulin (Ig) G in serum samples and IgA in nasal and lung lavage fluids were analyzed using enzyme-linked immunosorbent assay. Cytokine release and proliferation capacity of splenic lymphocytes in response to antigens were measured in vitro. The protective effect of the C-HapS-P6 protein against NTHi infection was evaluated by NTHi count and histological examination. The data showed that the C-HapS-P6 fusion protein increased significantly the levels of serum IgG and nasal and lung IgA, and promoted the release of interleukin (IL)-2, interferon-ϒ, IL-4, IL-5, and IL-17 and the proliferation of splenic lymphocytes compared with C-HapS or P6 protein treatment alone. Moreover, C-HapS-P6 effectively reduced the NTHi colonization in the nasopharynx and lungs of mice. In conclusion, our results demonstrated that the C-HapS-P6 fusion protein vaccine can significantly enhance humoral and cell immune responses and effectively prevent against NTHi infection in the respiratory tract in murine models.

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.

Enhancing sensitivity of qPCR assays targeting Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae by using a mutant Taq DNA polymerase.

AIMS: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae are important causes of bacterial meningitis. In this study, the DNA binding site of the wild type Taq DNA polymerase was modified to produce a mutant enzyme with enhanced DNA affinity and PCR performance. The engineered and the wild type enzymes were integrated into qPCR-based assays for molecular detection of S. pneumoniae, N. meningitidis, H. influenzae, and serogroups and serotypes of these three pathogens. METHODS: Bio-Speedy® Bacterial DNA Isolation Kit (Bioeksen R&D Technologies, Turkiye) and 2× qPCR-Mix for hydrolysis probes (Bioeksen R&D Technologies, Turkiye) and CFX96 Instrument (Biorad Inc., USA) were used for all molecular analyses. Spiked negative clinical specimens were tested using the developed qPCR assays and the culture-based conventional methods for the analytical performance evaluation. RESULTS: All qPCR assays did not produce any positive results for the samples spiked with potential cross-reacting bacteria. Limit of detection (LOD) of the assays containing the mutant enzyme was 1 genome/reaction (10 cfu/mL sample) which is at least 3 times lower than the previously reported LOD levels for DNA amplification based molecular assays. LODs for the spiked serum and cerebrospinal fluid (CSF) samples decreased 2.3-4.7 and 1.2-3.5 times respectively when the mutant enzyme was used instead of the wild type Taq DNA polymerase. CONCLUSIONS: It is possible to enhance analytical sensitivity of qPCR assays targeting the bacterial agents of meningitis by using an engineered Taq DNA polymerase. These qPCR-based assays can be used for direct detection and serogrouping / serotyping of S. pneumoniae, N. meningitidis and H. influenzae at concentrations close to the lower limit of medical decision point.

Rare serotype c Haemophilus influenzae invasive isolate: characterization of the first case in Portugal.

We report for the first time in Portugal a serotype c Haemophilus influenzae isolated from an adult, with HIV-1 infection. Whole-genome sequencing characterized the isolate as clonal complex ST-7, albeit with a novel MLST (ST2754) due to a unique atpG profile. Integration of this genome with other available H. influenzae serotype c genomes from PubMLST revealed its overall genetic distinctiveness, with the closest related isolate being identified in France in 2020. This surveillance study, involving collaboration among hospitals and reference laboratory, successfully contributed to the identification and characterization of this rare serotype.

Hfe Permease and Haemophilus influenzae Manganese Homeostasis.

Haemophilus influenzae is a commensal of the human upper respiratory tract that can infect diverse host niches due, at least in part, to its ability to withstand both endogenous and host-mediated oxidative stresses. Here, we show that hfeA, a gene previously linked to iron import, is essential for H. influenzae manganese recruitment via the HfeBCD transporter. Structural analyses show that metal binding in HfeA uses a unique mechanism that involves substantial rotation of the C-terminal lobe of the protein. Disruption of hfeA reduced H. influenzae manganese acquisition and was associated with decreased growth under aerobic conditions, impaired manganese-superoxide dismutase activity, reduced survival in macrophages, and changes in biofilm production in the presence of superoxide. Collectively, this work shows that HfeA contributes to H. influenzae manganese acquisition and virulence attributes. High conservation of the hfeABCD permease in Haemophilus species suggests that it may serve similar roles in other pathogenic Pasteurellaceae.

Phasevarions in Haemophilus influenzae biogroup aegyptius control expression of multiple proteins.

IMPORTANCE: Haemophilus influenzae biogroup aegyptius is a human-adapted pathogen and the causative agent of Brazilian purpuric fever (BPF), an invasive disease with high mortality, that sporadically manifests in children previously suffering conjunctivitis. Phase variation is a rapid and reversible switching of gene expression found in many bacterial species, and typically associated with outer-membrane proteins. Phase variation of cytoplasmic DNA methyltransferases has been shown to play important roles in bacterial gene regulation and can act as epigenetic switches, regulating the expression of multiple genes as part of systems called phasevarions (phase-variable regulons). This study characterized two alleles of the ModA phasevarion present in H. influenzae biogroup aegyptius, ModA13, found in non-BPF causing strains and ModA16, unique to BPF causing isolates. Phase variation of ModA13 and ModA16 led to genome-wide changes to DNA methylation resulting in altered protein expression. These changes did not affect serum resistance in H. influenzae biogroup aegyptius strains.

The Association between Transformation Ability and Antimicrobial Resistant Potential in Haemophilus influenzae.

The prevalence of quinolone low-susceptible Haemophilus influenzae has increased in Japan. Low quinolone susceptibility is caused by point mutations in target genes; however, it can also be caused by horizontal gene transfer via natural transformation. In this study, we examined whether this horizontal gene transfer could be associated with resistance to not only quinolones but also other antimicrobial agents. Horizontal transfer ability was quantified using the experimental transfer assay method for low quinolone susceptibility. Further, the association between horizontal transfer ability and resistance to ß-lactams, the first-choice drugs for H. influenzae infection, was investigated. The transformation efficiency of 50 clinical isolates varied widely, ranging from 102 to 106 colony forming unit (CFU) of the colonies obtained by horizontal transfer assay. Efficiency was associated with ß-lactam resistance caused by ftsI mutations, indicating that strains with high horizontal transfer ability acquired quinolone low-susceptibility as well as ß-lactam resistance more easily. Strains with high transformation efficiency increased the transcript level of comA, suggesting that enhanced com operon was associated with a high DNA uptake ability. Overall, this study revealed that the transformation ability of H. influenzae was associated with multiple antimicrobial resistance. Increase in the number of strains with high horizontal transformation ability has raised concerns regarding the rapid spread of antimicrobial-resistant H. influenzae.

Biofilm Formation by Nontypeable Haemophilus Influenzae and Resistance to Complement-Mediated Clearance.

Biofilm formation has been suggested to be associated with phenotype changes compared with the planktonic form. We screened 1092 Haemophilus influenzae isolates for their genetic relationships and then selected 29 isolates from different genotypes and phenotypes and tested their ability to form biofilm. Our data showed a higher capacity of nontypable isolates, particularly isolates from respiratory and genital infections to form biofilm, compared with typable isolates. This ability to form biofilm was also correlated with reduced deposition of the complement component C3b on biofilm-involved bacteria. These data suggest that the biofilm formation contributes to the virulence of nontypable H. influenzae.

Informed interpretation of metagenomic data by StrainPhlAn enables strain retention analyses of the upper airway microbiome.

IMPORTANCE: The usage of 16S rRNA gene sequencing has become the state-of-the-art method for the characterization of the microbiota in health and respiratory disease. The method is reliable for low biomass samples due to prior amplification of the 16S rRNA gene but has limitations as species and certainly strain identification is not possible. However, the usage of metagenomic tools for the analyses of microbiome data from low biomass samples is not straight forward, and careful optimization is needed. In this work, we show that by validating StrainPhlAn 3 results with the data from bacterial cultures, the strain-level tracking of the respiratory microbiome is feasible despite the high content of host DNA being present when parameters are carefully optimized to fit low biomass microbiomes. This work further proposes that strain retention analyses are feasible, at least for more abundant species. This will help to better understand the longitudinal dynamics of the upper respiratory microbiome during health and disease.

Molecular epidemiology and antimicrobial resistance of Haemophilus influenzae isolates from otitis media in Bulgaria.

Haemophilus influenzae is one of the main bacteria responsible for otitis media (OM) among children worldwide. We aimed to estimate the distribution of encapsulated and non-capsulated variants (NTHi), biotypes, antibiotic susceptibility, and molecular epidemiology of H. influenzae isolates recovered from pediatric OM cases in Bulgaria.Capsule detection was done by PCR for bexB gene, absent in NTHi. All encapsulated strains were subjected to PCR serotyping. MIC susceptibility testing was performed according to the criteria of EUCAST. MLST was conducted for all 71 OM isolates.The capsule detection and PCR - serotyping disclosed a predominance of NTHi (90.1%) and a few "a", "f", and "c" types. Biotype I was the most widespread (42.3%). ß-lactam resistance was found in 35.2% of the isolates. MLST represented heterogenic population structure, whereas the most represented clonal complexes belonged to ST-3, ST-57, ST-105, and ST-1426. 42.3% of the STs showed relatedness to globally represented clones, and 11.3% displayed affiliation to international type 2.Most of the H. influenzae isolates recovered from children with otitis media were non-typable strains from biotype I. The examined population structure was genetically diverse, with a predominance of international type 2 isolates.