Impact of the COVID-19 pandemic on Haemophilus influenzae infections in pediatric patients hospitalized with community acquired pneumonia.

The COVID-19 pandemic has altered the infection landscape for many pathogens. This retrospective study aimed to compare Haemophilus influenzae (H. influenzae) infections in pediatric CAP patients hospitalized before (2018-2019) and during (2020-2022) the COVID-19 pandemic. We analyzed the clinical epidemiology and antimicrobial resistance (AMR) patterns of H. influenzae from a tertiary hospital in southwest China. A total of 986 pediatric CAP patients with H. influenzae-associated infections were included. Compared to 2018, the positivity rate increased in 2019 but dropped significantly in 2020. Although it rose in the following 2 years, the rate in 2022 remained significantly lower than in 2019. Patients' age during the pandemic was significantly higher than in 2018 and 2019, while gender composition remained similar across both periods. Notably, there were significant changes in co-infections with several respiratory pathogens during the pandemic. Resistance rates of H. influenzae isolates to antibiotics varied, with the highest resistance observed for ampicillin (85.9%) and the lowest for cefotaxime (0.0%). Resistance profiles to various antibiotics underwent dramatic changes during the COVID-19 pandemic. Resistance to amoxicillin-clavulanate, cefaclor, cefuroxime, trimethoprim-sulfamethoxazole, and the proportion of multi-drug resistant (MDR) isolates significantly decreased. Additionally, MDR isolates, alongside isolates resistant to specific drugs, were notably prevalent in ampicillin-resistant and ß-lactamase-positive isolates. The number of pediatric CAP patients, H. influenzae infections, and isolates resistant to certain antibiotics exhibited seasonal patterns, peaking in the winter of 2018 and 2019. During the COVID-19 pandemic, sharp decreases were observed in February 2020, and there was no resurgence in December 2022. These findings indicate that the COVID-19 pandemic has significantly altered the infection spectrum of H. influenzae in pediatric CAP patients, as evidenced by shifts in positivity rate, demographic characteristics, respiratory co-infections, AMR patterns, and seasonal trends.

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.

Inhaled antibiotics: A promising drug delivery strategies for efficient treatment of lower respiratory tract infections (LRTIs) associated with antibiotic resistant biofilm-dwelling and intracellular bacterial pathogens.

Antibiotic-resistant bacteria associated with LRTIs are frequently associated with inefficient treatment outcomes. Antibiotic-resistant Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, and Staphylococcus aureus, infections are strongly associated with pulmonary exacerbations and require frequent hospital admissions, usually following failed management in the community. These bacteria are difficult to treat as they demonstrate multiple adaptational mechanisms including biofilm formation to resist antibiotic threats. Currently, many patients with the genetic disease cystic fibrosis (CF), non-CF bronchiectasis (NCFB) and chronic obstructive pulmonary disease (COPD) experience exacerbations of their lung disease and require high doses of systemically administered antibiotics to achieve meaningful clinical effects, but even with high systemic doses penetration of antibiotic into the site of infection within the lung is suboptimal. Pulmonary drug delivery technology that reliably deliver antibacterials directly into the infected cells of the lungs and penetrate bacterial biofilms to provide therapeutic doses with a greatly reduced risk of systemic adverse effects. Inhaled liposomal-packaged antibiotic with biofilm-dissolving drugs offer the opportunity for targeted, and highly effective antibacterial therapeutics in the lungs. Although the challenges with development of some inhaled antibiotics and their clinicals trials have been studied; however, only few inhaled products are available on market. This review addresses the current treatment challenges of antibiotic-resistant bacteria in the lung with some clinical outcomes and provides future directions with innovative ideas on new inhaled formulations and delivery technology that promise enhanced killing of antibiotic-resistant biofilm-dwelling bacteria.

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.

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.

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.

Antimicrobial Susceptibility Survey of Invasive Haemophilus influenzae in the United States in 2016.

Antibiotics are important for the treatment and prevention of invasive Haemophilus influenzae disease. Reduced susceptibility to clinically relevant drugs, except ampicillin, has been uncommon in the United States. Susceptibility of 700 invasive H. influenzae isolates, collected through population-based surveillance during 2016, was assessed for 15 antibiotics using broth microdilution, according to the CLSI guidelines; a subset of 104 isolates were also assessed for rifampin susceptibility using Etest. Genomes were sequenced to identify genes and mutations known to be associated with reduced susceptibility to clinically relevant drugs. A total of 508 (72.6%) had reduced susceptibility to at least one antibiotic and more than half of the isolates exhibited reduced susceptibility to only one (33.6%) or two (21.6%) antibiotic classes. All tested isolates were susceptible to rifampin, a chemoprophylaxis agent, and <1% (n = 3) of isolates had reduced susceptibility to third generation cephalosporins, which are recommended for invasive disease treatment. In contrast, ampicillin resistance was more common (28.1%) and predominantly associated with the detection of a ß-lactamase gene; 26.2% of isolates in the collection contained either a TEM-1 or ROB-1 ß-lactamase gene, including 88.8% of ampicillin-resistant isolates. ß-lactamase negative ampicillin-resistant (BLNAR) isolates were less common and associated with ftsI mutations; resistance to amoxicillin-clavulanate was detected in <2% (n = 13) of isolates. The proportion of reduced susceptibility observed was higher among nontypeable H. influenzae and serotype e than other serotypes. US invasive H. influenzae isolates remain predominantly susceptible to clinically relevant antibiotics except ampicillin, and BLNAR isolates remain uncommon. IMPORTANCE Antibiotics play an important role for the treatment and prevention of invasive Haemophilus influenzae disease. Antimicrobial resistance survey of invasive H. influenzae isolates collected in 2016 showed that the US H. influenzae population remained susceptible to clinically relevant antibiotics, except for ampicillin. Detection of approximately a quarter ampicillin-resistant and ß-lactamase containing strains demonstrates that resistance mechanisms can be acquired and sustained within the H. influenzae population, highlighting the continued importance of antimicrobial resistance surveillance for H. influenzae to monitor susceptibility trends and mechanisms of resistance.

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.

Impact of the introduction of EUCAST's concept of "area of technical uncertainty".

On the first of January 2019, the European Committee on Antimicrobial Susceptibility Testing, EUCAST, introduced the concept of "area of technical uncertainty" (ATU). The aim was to report on the incidence of ATU test results in a selection of common bacterial species and the subsequent impact on antimicrobial resistance categorization and workload. A retrospective analysis of clinical samples collected from February 2019 until November 2019 was performed. Susceptibility to amoxicillin-clavulanic acid and piperacillin-tazobactam in Enterobacterales (Escherichia spp., Klebsiella spp., Proteus spp.), piperacillin-tazobactam in Pseudomonas aeruginosa, and amoxicillin-clavulanic acid and cefuroxime in Haemophilus influenzae was studied. Disk diffusion antibiotic susceptibility testing was read and interpreted by ADAGIO 93400 automated system (Bio-Rad, France). In case of an inhibition zone in the ATU, strains were retested using gradient minimal inhibitory concentration method (Etest, BioMérieux, France). Overall, 14,164 isolate-antibiotic combinations were tested in 7922 isolates, resulting in 1204 (8.5%) disk zone diameters in the ATU region. Retesting of ATUs with Etest resulted in a category change from S to R for amoxicillin-clavulanic acid in 63/498 (12.7%) of Escherichia spp., 2/58 (3.4%) of Klebsiella spp., 2/37 (5.4%) of Proteus spp., and 6/125 (4.8%) of Haemophilus influenzae. For piperacillin-tazobactam, a category change from S to R was found in 33/92 (35.9%) of Pseudomonas aeruginosa. We conclude that ATU testing has a substantial impact on the correct interpretation of antimicrobial resistance, at the expense of turn-around time and with the cost of additional workload.

Selective Capture of Anti-N-glucosylated NTHi Adhesin Peptide Antibodies by a Multivalent Dextran Conjugate.

Tentacle-like polymers decorated with several copies of peptide antigens can be interesting tools for increasing the ability to capture circulating antibodies in patient sera, using cooperative effects for stronger avidity. We previously showed that antibodies from multiple sclerosis (MS) patient sera preferentially recognize hyperglucosylated adhesin protein HMW1ct of non-typeable Haemophilus influenzae (NTHi). We selected the C-terminal HMW1ct(1347-1354) minimal epitope and prepared the diglucosylated analogue Ac-KAN(Glc)VTLN(Glc)TTG-K(N3 )-NH2 to graft a 40 kDa dextran scaffold modified with glycidyl-propargyl moieties to perform a copper catalyzed alkyne-azide coupling reaction (CuAAC). Quantitative NMR measurements allowed the characterization of the peptide loading (19.5 %) on the multivalent dextran conjugate. This novel polymeric structure displayed optimal capturing properties of both IgG and, more interestingly, IgM antibodies in MS sera. Specific antibodies from a representative MS serum, were successfully depleted using a Sepharose resin bearing the new glucosylated multivalent conjugate, as confirmed by ELISA. These results may offer a promising proof-of-concept for the selective purification of high affinity autoantibodies from sera of autoimmune patients, in general, and of specific high affinity antibodies against a minimally glcosylated epitope Asn(Glc) from sera of multiple sclerosis (MS) patients, in particular.

Multiplex polymerase chain reaction detection of Streptococcus pneumoniae and Haemophilus influenzae and their antibiotic resistance in patients with community-acquired pneumonia from southwest Iran.

BACKGROUND: This study aimed to evaluate the occurrence of Streptococcus pneumoniae and Haemophilus influenzae in sputum of patients with community-acquired pneumonia (CAP) using culture and multiplex polymerase chain reaction (M-PCR) methods and to survey the antibiotic resistance patterns of aforesaid isolates. RESULT: In total, 23.9 % (n = 22/92) of sputum samples showed positive results in the culture method. S. pneumoniae and H. influenzae were isolated from 15 (16.3 %) and 7 (7.6%) samples, respectively. Using M-PCR, 44 (47.8 %) samples were positive for S. pneumoniae and H. influenzae. Of these, S. pneumoniae and H. influenzae were detected in 33 (35.8%) and 11 (11.9%) of the sputum samples, respectively. The sensitivity, specificity, and accuracy rates of PCR in detection of S. pneumoniae in comparison with culture method were 100, 76.6, and 83.6%, respectively. While, the sensitivity, specificity, and accuracy rates of PCR in detection of H. influenzae in comparison with culture method were 100, 95.3, and 95.8%, respectively. Out of 11 isolates of H. influenzae, two strains confirmed as H. influenzae type b (Hib) and 3 isolates were type f. However, 6 isolates were non-typable. The co-trimoxazole and amoxicillin/clavulanate were the less effective antibiotics against S. pneumonia and H. influenzae, respectively. Ceftriaxone with 13.3% resistance rates was the most effective antibiotic against S. pneumoniae, while, clarithromycin, ceftriaxone, and gentamicin with resistance rates of 28.6% for each one were the most effective chemicals against H. influenzae isolates. CONCLUSION: In this study, the prevalence of S. pneumoniae was more than H. influenzae using culture and M-PCR methods. The M-PCR provided better efficiency in detecting the bacterial agents in CAP patients compared to culture method. This method can improve the early detection of pathogens contributed to CAP. The drug resistant S. pneumoniae and H. influenzae indicated the need to develop a codified monitoring program to prevent further spread of these strains.

Antibacterial Activity of Thymus vulgaris L. Essential Oil Vapours and Their GC/MS Analysis Using Solid-Phase Microextraction and Syringe Headspace Sampling Techniques.

While the inhalation of Thymus vulgaris L. essential oil (EO) is commonly approved for the treatment of mild respiratory infections, there is still a lack of data regarding the antimicrobial activity and chemical composition of its vapours. The antibacterial activity of the three T. vulgaris EOs against respiratory pathogens, including Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pyogenes, was assessed in both liquid and vapour phases using the broth microdilution volatilisation (BMV) method. With the aim of optimising a protocol for the characterisation of EO vapours, their chemical profiles were determined using two headspace sampling techniques coupled with GC/MS: solid-phase microextraction (HS-SPME) and syringe headspace sampling technique (HS-GTS). All EO sample vapours exhibited antibacterial activity with minimum inhibitory concentrations (MIC) ranging from 512 to 1024 µg/mL. According to the sampling technique used, results showed a different distribution of volatile compounds. Notably, thymol was found in lower amounts in the headspace-peak percentage areas below 5.27% (HS-SPME) and 0.60% (HS-GTS)-than in EOs (max. 48.65%), suggesting that its antimicrobial effect is higher in vapour. Furthermore, both headspace sampling techniques were proved to be complementary for the analysis of EO vapours, whereas HS-SPME yielded more accurate qualitative results and HS-GTS proved a better technique for quantitative analysis.

The structure of nontypeable Haemophilus influenzae SapA in a closed conformation reveals a constricted ligand-binding cavity and a novel RNA binding motif.

Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in respiratory disease and otitis media. Important for NTHi survival, colonization and persistence in vivo is the Sap (sensitivity to antimicrobial peptides) ABC transporter system. Current models propose a direct role for Sap in heme and antimicrobial peptide (AMP) transport. Here, the crystal structure of SapA, the periplasmic component of Sap, in a closed, ligand bound conformation, is presented. Phylogenetic and cavity volume analysis predicts that the small, hydrophobic SapA central ligand binding cavity is most likely occupied by a hydrophobic di- or tri- peptide. The cavity is of insufficient volume to accommodate heme or folded AMPs. Crystal structures of SapA have identified surface interactions with heme and dsRNA. Heme binds SapA weakly (Kd 282 µM) through a surface exposed histidine, while the dsRNA is coordinated via residues which constitute part of a conserved motif (estimated Kd 4.4 µM). The RNA affinity falls within the range observed for characterized RNA/protein complexes. Overall, we describe in molecular-detail the interactions of SapA with heme and dsRNA and propose a role for SapA in the transport of di- or tri-peptides.

The effects of the dietary compound L-sulforaphane against respiratory pathogens.

L-sulforaphane (LSF) is an isothiocyanate derived from cruciferous vegetables that has long been known for its anticarcinogenic, antioxidant and anti-inflammatory effects. LSF also possesses antimicrobial properties, although the evidence for this is limited. Respiratory pathogens, such as Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus pyogenes and respiratory syncytial virus (RSV), are leading global causes of illness and death among children aged under five years, particularly in resource-poor countries where access to vaccines are limited or, in the case of S. pyogenes and RSV, vaccines have not been licensed for use in humans. Therefore, alternative strategies to prevent and/or treat these common infectious diseases are urgently needed. This study was conducted to investigate the antimicrobial effects of LSF against common respiratory pathogens, S. pneumoniae (serotypes 1 and 6B), H. influenzae type B (HiB), non-typeable H. influenzae (NTHi), S. pyogenes and RSV in relevant human cell-based models. LSF significantly inhibited the growth of H. influenzae, but not S. pneumoniae or S. pyogenes. LSF did not improve opsonophagocytic capacity or killing by human phagocytic cell lines (HL-60s and THP-1 macrophages) for S. pneumoniae yet showed some improved killing for H. influenzae species in THP-1 macrophages. However, LSF significantly reduced RSV infection in human lung epithelial cells, associated with increased expression of cyclin D1 (CCND1) gene as well as the antioxidant genes, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HMOX-1). Overall, LSF represents an exciting avenue for further antimicrobial research, particularly as a novel therapy against H. influenzae species and RSV.

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.

Antimicrobial resistance of Haemophilus influenzae isolates from pediatric hospitals in Mainland China: Report from the ISPED program, 2017-2019.

PURPOSE: This study set out to determine the antimicrobial resistance trends of Haemophilus influenzae isolates from pediatric hospitals in Mainland China, which would provide basis for clinical treatment. METHODS: The Infectious Disease Surveillance of Pediatrics (ISPED) collaboration group conducted this study. H. influenzae strains isolated from nine pediatric hospitals in Mainland China were included. Disk diffusion method was used for antimicrobial susceptibility test. Cefinase disc was used for detection of ß-lactamase. RESULTS: In total, 13810 H. influenzae isolates were included during 2017-2019: 93.17% of which were from respiratory tract specimens, 4.63% from vaginal swabs, 1.10% from secretion, and 1.10% from others. Of all strains, 63.32% isolates produced ß-lactamase; 8.22% isolates were ß-lactamase-negative and ampicillin-resistant (BLNAR). The resistance to sulfamethoxazole-trimethoprim was 70.98%, followed by resistance to ampicillin (69.37%), cefuroxime (51.35%), ampicillin-sulbactam (38.82%), azithromycin (38.21%), amoxicillin-clavulanate (35.28%). More than 90% of H. influenzae isolates were susceptible to ceftriaxone, cefotaxime, meropenem, levofloxacin and chloramphenicol. The resistance rate of ampicillin and azithromycin in H. influenzae showed an increasing trend through the years. Statistically significant differences in antibiotic-resistance rates of all the antibiotics except chloramphenicol were found in different regions. The major Multi-Drug Resistance pattern was resistant to ß-lactams, macrolides, and sulfonamides. CONCLUSIONS: There is a rising trend of resistance rate of ampicillin and azithromycin in H. influenzae. Antimicrobial resistance of H. influenzae deserves our ongoing attention. Third-generation cephalosporin could be the preferred treatment option of infections caused by ampicillin-resistant H. influenzae.

Antimicrobial Susceptibility among Gram-Negative Isolates in Pediatric Patients in Europe from 2013-2018 Compared to 2004-2012: Results from the ATLAS Surveillance Study.

AIMS: Data on antimicrobial resistance (AMR) in the paediatric patient population are scarce. This study assessed the AMR rates and phenotype distribution of Gram-negative isolates in paediatric patients in Europe from 2004-2012 and 2013-2018. METHODS: Isolates that were collected were stratified by age groups (< 1, 1-5, 6-12, and 13-17 years) and regions (North-Western, Eastern and Southern Europe). Minimal inhibitory concentrations (broth microdilution) were interpreted according to European Committee on Antimicrobial Susceptibility Testing guidelines. Resistance rates and phenotype prevalence were identified for Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Haemophilus influenzae. RESULTS: In the overall paediatric patient population (0-17 years), extended-spectrum beta-lactamase (ESBL) production significantly decreased (from 20.7% to 15.4%, P < 0.0001) in Escherichia coli, whereas it increased for Klebsiella pneumoniae (from 35.0% to 39.2%, P = 0.015). Carbapenem resistance was highest for Acinetobacter baumannii (32.3%) compared with Klebsiella pneumoniae (4.7%) and Pseudomonas aeruginosa (12.4%) in 2013-2018, and rates were significantly increased relative to 2004-2012. There was no change in resistance to beta-lactam antimicrobials for Haemophilus influenzae. The lowest resistance rates for most organism groups were observed in North-Western Europe. CONCLUSIONS: The results revealed a significant increase in Klebsiella pneumoniae isolates with an ESBL and carbapenem-resistance phenotype as well as in carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa from 2004-2012 to 2013-2018. Conversely, a decrease in ESBL E. coli was observed. Continued surveillance and awareness of resistance in these bacteria causing serious infections is crucial for improving treatment quality in paediatric patients.

New Broth Macrodilution Volatilization Method for Antibacterial Susceptibility Testing of Volatile Agents and Evaluation of Their Toxicity Using Modified MTT Assay In Vitro.

In this study, a new broth macrodilution volatilization method for the simple and rapid determination of the antibacterial effect of volatile agents simultaneously in the liquid and vapor phase was designed with the aim to assess their therapeutic potential for the development of new inhalation preparations. The antibacterial activity of plant volatiles (ß-thujaplicin, thymohydroquinone, thymoquinone) was evaluated against bacteria associated with respiratory infections (Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes) and their cytotoxicity was determined using a modified thiazolyl blue tetrazolium bromide assay against normal lung fibroblasts. Thymohydroquinone and thymoquinone possessed the highest antibacterial activity against H. influenzae, with minimum inhibitory concentrations of 4 and 8 µg/mL in the liquid and vapor phases, respectively. Although all compounds exhibited cytotoxic effects on lung cells, therapeutic indices (TIs) suggested their potential use in the treatment of respiratory infections, which was especially evident for thymohydroquinone (TI > 34.13). The results demonstrate the applicability of the broth macrodilution volatilization assay, which combines the principles of broth microdilution volatilization and standard broth macrodilution methods. This assay enables rapid, simple, cost- and labor-effective screening of volatile compounds and overcomes the limitations of assays currently used for screening of antimicrobial activity in the vapor phase.

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.

Comparison of the bactericidal effects of quinolones against low-susceptible Haemophilus influenzae.

The increasing incidence of Haemophilus influenzae with decreased susceptibility to quinolones (quinolone low-susceptible H. influenzae) in Japan has raised concerns about therapeutic failure. Thus, assessment of effective antimicrobial agents is necessary to establish an effective therapeutic strategy against resulting infections. In this study, in vitro bactericidal effects of quinolones on low-susceptible H. influenzae strains were evaluated using time-kill curve analysis. For tosufloxacin, log reduction values for low-susceptible strains were significantly lower than those for susceptible strains at both Cmax and 1/2 Cmax. Conversely, although the log reduction values were lower for susceptible strains, the Cmax of levofloxacin and ß-lactams (amoxicillin and cefditoren) indicated bactericidal effects. In addition, higher concentrations of tosufloxacin at 2×Cmax and 4×Cmax had bactericidal effects on not only susceptible but also low-susceptible strains. These data strongly suggest that we should consider the presence of low-susceptible strains and reconsider the appropriate dosage of tosufloxacin for treatment, especially for paediatric patients.