High-resolution genomics identifies pneumococcal diversity and persistence of vaccine types in children with community-acquired pneumonia in the UK and Ireland.

BACKGROUND: Streptococcus pneumoniae is a global cause of community-acquired pneumonia (CAP) and invasive disease in children. The CAP-IT trial (grant No. 13/88/11; https://www.capitstudy.org.uk/ ) collected nasopharyngeal swabs from children discharged from hospitals with clinically diagnosed CAP, and found no differences in pneumococci susceptibility between higher and lower antibiotic doses and shorter and longer durations of oral amoxicillin treatment. Here, we studied in-depth the genomic epidemiology of pneumococcal (vaccine) serotypes and their antibiotic resistance profiles. METHODS: Three-hundred and ninety pneumococci cultured from 1132 nasopharyngeal swabs from 718 children were whole-genome sequenced (Illumina) and tested for susceptibility to penicillin and amoxicillin. Genome heterogeneity analysis was performed using long-read sequenced isolates (PacBio, n = 10) and publicly available sequences. RESULTS: Among 390 unique pneumococcal isolates, serotypes 15B/C, 11 A, 15 A and 23B1 were most prevalent (n = 145, 37.2%). PCV13 serotypes 3, 19A, and 19F were also identified (n = 25, 6.4%). STs associated with 19A and 19F demonstrated high genome variability, in contrast to serotype 3 (n = 13, 3.3%) that remained highly stable over a 20-year period. Non-susceptibility to penicillin (n = 61, 15.6%) and amoxicillin (n = 10, 2.6%) was low among the pneumococci analysed here and was independent of treatment dosage and duration. However, all 23B1 isolates (n = 27, 6.9%) were penicillin non-susceptible. This serotype was also identified in ST177, which is historically associated with the PCV13 serotype 19F and penicillin susceptibility, indicating a potential capsule-switch event. CONCLUSIONS: Our data suggest that amoxicillin use does not drive pneumococcal serotype prevalence among children in the UK, and prompts consideration of PCVs with additional serotype coverage that are likely to further decrease CAP in this target population. Genotype 23B1 represents the convergence of a non-vaccine genotype with penicillin non-susceptibility and might provide a persistence strategy for ST types historically associated with vaccine serotypes. This highlights the need for continued genomic surveillance.

Increase in bloodstream infections caused by emm1 group A Streptococcus correlates with emergence of toxigenic M1UK, Belgium, May 2022 to August 2023.

Many European countries have recently reported upsurges in invasive group A Streptococcus (iGAS) infections, mainly caused by emm1 Streptococcus pyogenes, specifically the toxigenic M1UK lineage. We present the epidemiology of emm1 causing iGAS in Belgium during 2018-August 2023, and describe an emergence of the toxigenic M1UK lineage in Belgium in mid-2022 that was observed as an increase in bloodstream infections caused by emm1 S. pyogenes that continued into 2023.

Haemophilus influenzae carriage and antibiotic resistance profile in Belgian infants over a three-year period (2016-2018).

Background: Non-typeable Haemophilus influenzae has become increasingly important as a causative agent of invasive diseases following vaccination against H. influenzae type b. The emergence of antibiotic resistance underscores the necessity to investigate typeable non-b carriage and non-typeable H. influenzae (NTHi) in children. Methods: Nasopharyngeal swab samples were taken over a three-year period (2016-2018) from 336 children (6-30 months of age) attending daycare centers (DCCs) in Belgium, and from 218 children with acute otitis media (AOM). Biotype, serotype, and antibiotic resistance of H. influenzae strains were determined phenotypically. Mutations in the ftsI gene were explored in 129 strains that were resistant or had reduced susceptibility to beta-lactam antibiotics. Results were compared with data obtained during overlapping time periods from 94 children experiencing invasive disease. Results: Overall, NTHi was most frequently present in both carriage (DCC, AOM) and invasive group. This was followed by serotype "f" (2.2%) and "e" (1.4%) in carriage, and "b" (16.0%), "f" (11.7%), and "a" (4.3%) in invasive strains. Biotype II was most prevalent in all studied groups, followed by biotype III in carriage and I in invasive strains. Strains from both groups showed highest resistance to ampicillin (26.7% in carriage vs. 18.1% in invasive group). A higher frequency of ftsI mutations were found in the AOM group than the DCC group (21.6 vs. 14.9% - p = 0.056). Even more so, the proportion of biotype III strains that carried a ftsI mutation was higher in AOM compared to DCC (50.0 vs. 26.3% - p < 0.01) and invasive group. Conclusion: In both groups, NTHi was most frequently circulating, while specific encapsulated serotypes for carriage and invasive group were found. Biotypes I, II and III were more frequently present in the carriage and invasive group. The carriage group had a higher resistance-frequency to the analyzed antibiotics than the invasive group. Interestingly, a higher degree of ftsI mutations was found in children with AOM compared to DCC and invasive group. This data helps understanding the H. influenzae carriage in Belgian children, as such information is scarce.

Host Immunity Influences the Composition of Murine Gut Microbiota.

The influence of gut microbiota on host immunity is widely studied, and its disturbance has been linked to several immune-mediated disorders. Conversely, whether and how inherently disturbed canonical Th1 (pro-inflammatory) and/or Th2 (anti-inflammatory) immune pathways modify the host microbiome is not sufficiently investigated. Here, we characterized the humoral, cellular, and cytokine immunity, and associated alterations in gut microbiota of naïve wild-type mice (C57BL/6 and BALB/c), and mice with deficiencies in Th2 responses (IL-4Rα and IL-33 knockout mice) or in both Th1 and Th2 responses (NOD scid gamma, NSG mice). A global analysis by de novo clustering of 16S rRNA profiles of the gut microbiota independently grouped wild-type immunocompetent (C57BL/6 and BALB/c), Th2-deficient (IL-4Rα-/- and IL-33-/-), and severely immunodeficient (NSG) mice; where wild-type mice, but not Th2 or severely immunodeficient mice, were enriched in gut bacteria that produce short-chain fatty acids. These include members of phyla Firmicutes, Verrucomicrobia, and Bacteroidetes such as Lactobacillus spp., Akkermansia muciniphila, and Odoribacter spp. Further comparison of the two naïve wild-type mouse strains showed higher microbial diversity (Shannon), primarily linked to higher richness (Chao1), as well as a distinct difference in microbial composition (weighted UniFrac) in BALB/c mice compared to C57BL/6. T-cell and blood cytokine analyses demonstrated a Th1-polarization in naïve adaptive immunity in C57BL/6 animals compared to BALB/c mice, and an expected Th2 deficient cellular response in IL-4Rα-/- and IL-33-/- mice compared to its genetic background BALB/c strain. Together, these data suggest that alterations in the Th1/Th2 balance or a complete ablation of Th1/Th2 responses can lead to major alterations in gut microbiota composition and function. Given the similarities between the human and mouse immune systems and gut microbiota, our finding that immune status is a strong driver of gut microbiota composition has important consequences for human immunodeficiency studies.

Diversity in the Characteristics of Klebsiella pneumoniae ST101 of Human, Environmental, and Animal Origin.

BACKGROUND: Klebsiella pneumoniae ST101 is an emerging high-risk clone which exhibits extensive drug resistance. Bacterial strains residing in multiple hosts show unique signatures related to host adaptation. In this study, we assess the genetic relationship of K. pneumoniae ST101 isolated from hospital samples, the environment, community, and livestock using whole genome sequencing (WGS). MATERIALS AND METHODS: We selected ten K. pneumoniae ST101 strains from hospitalized patients in Italy (n = 3) (2014) and Spain (n = 5) (2015-2016) as well as Belgian livestock animals (n = 2) (2017-2018). WGS was performed with 2 × 250 bp paired-end sequencing (Nextera XT) sample preparation kit and MiSeq (Illumina Inc.). Long-read sequencing (Pacbio Sequel I) was used to sequence the two livestock strains and three Italian hospital-associated strains. Furthermore, a public ST101 sequence collection of 586 strains (566 hospital-associated strains, 12 environmental strains, six strains from healthy individuals, one food-associated strain and one pig strain) was obtained. BacPipe and Kleborate were used to conduct genome analysis. ISFinder was used to find IS elements, and PHASTER was utilized to identify prophages. A phylogenetic tree was constructed to illustrate genetic relatedness. RESULTS: Hospital-associated K. pneumoniae ST101 showed higher resistance scores than non-clinical isolates from healthy individuals, the environment, food and livestock (1.85 ± 0.72 in hospital-associated isolates vs. 1.14 ± 1.13 in non-clinical isolates, p < 0.01). Importantly, the lack of integrative conjugative elements ICEKp bearing iron-scavenging yersiniabactin siderophores (ybt) in livestock-associated strains suggests a lower pathogenicity potential than hospital-associated strains. Mobile genetic elements (MGE) appear to be an important source of diversity in K. pneumoniae ST101 strains from different origins, with a highly stable genome and few recombination events outside the prophage-containing regions. Core genome MLST based analysis revealed a distinct genetic clustering between human and livestock-associated isolates. CONCLUSION: The study of K. pneumoniae ST101 hospital-associated and strains from healthy individuals and animals revealed a genetic diversity between these two groups, allowing us to identify the presence of yersiniabactin siderophores in hospital-associated isolates. Resistance and virulence levels in livestock-associated strains were considerably lower than hospital-associated strains, implying that the public health risk remains low. The introduction of an ICEKp into animal strains, on the other hand, might pose a public threat over time.

Effect of Amoxicillin Dose and Treatment Duration on the Need for Antibiotic Re-treatment in Children With Community-Acquired Pneumonia: The CAP-IT Randomized Clinical Trial.

Importance: The optimal dose and duration of oral amoxicillin for children with community-acquired pneumonia (CAP) are unclear. Objective: To determine whether lower-dose amoxicillin is noninferior to higher dose and whether 3-day treatment is noninferior to 7 days. Design, Setting, and Participants: Multicenter, randomized, 2 × 2 factorial noninferiority trial enrolling 824 children, aged 6 months and older, with clinically diagnosed CAP, treated with amoxicillin on discharge from emergency departments and inpatient wards of 28 hospitals in the UK and 1 in Ireland between February 2017 and April 2019, with last trial visit on May 21, 2019. Interventions: Children were randomized 1:1 to receive oral amoxicillin at a lower dose (35-50 mg/kg/d; n = 410) or higher dose (70-90 mg/kg/d; n = 404), for a shorter duration (3 days; n = 413) or a longer duration (7 days; n = 401). Main Outcomes and Measures: The primary outcome was clinically indicated antibiotic re-treatment for respiratory infection within 28 days after randomization. The noninferiority margin was 8%. Secondary outcomes included severity/duration of 9 parent-reported CAP symptoms, 3 antibiotic-related adverse events, and phenotypic resistance in colonizing Streptococcus pneumoniae isolates. Results: Of 824 participants randomized into 1 of the 4 groups, 814 received at least 1 dose of trial medication (median [IQR] age, 2.5 years [1.6-2.7]; 421 [52%] males and 393 [48%] females), and the primary outcome was available for 789 (97%). For lower vs higher dose, the primary outcome occurred in 12.6% with lower dose vs 12.4% with higher dose (difference, 0.2% [1-sided 95% CI -∞ to 4.0%]), and in 12.5% with 3-day treatment vs 12.5% with 7-day treatment (difference, 0.1% [1-sided 95% CI -∞ to 3.9]). Both groups demonstrated noninferiority with no significant interaction between dose and duration (P = .63). Of the 14 prespecified secondary end points, the only significant differences were 3-day vs 7-day treatment for cough duration (median 12 days vs 10 days; hazard ratio [HR], 1.2 [95% CI, 1.0 to 1.4]; P = .04) and sleep disturbed by cough (median, 4 days vs 4 days; HR, 1.2 [95% CI, 1.0 to 1.4]; P = .03). Among the subgroup of children with severe CAP, the primary end point occurred in 17.3% of lower-dose recipients vs 13.5% of higher-dose recipients (difference, 3.8% [1-sided 95% CI, -∞ to10%]; P value for interaction = .18) and in 16.0% with 3-day treatment vs 14.8% with 7-day treatment (difference, 1.2% [1-sided 95% CI, -∞ to 7.4%]; P value for interaction = .73). Conclusions and Relevance: Among children with CAP discharged from an emergency department or hospital ward (within 48 hours), lower-dose outpatient oral amoxicillin was noninferior to higher dose, and 3-day duration was noninferior to 7 days, with regard to need for antibiotic re-treatment. However, disease severity, treatment setting, prior antibiotics received, and acceptability of the noninferiority margin require consideration when interpreting the findings. Trial Registration: ISRCTN Identifier: ISRCTN76888927.

Study of the microbiological quality, prevalence of foodborne pathogens and product shelf-life of Gilthead sea bream (Sparus aurata) and Sea bass (Dicentrarchus labrax) from aquaculture in estuarine ecosystems of Andalusia (Spain).

This study was aimed at characterizing microbiologically Gilthead sea bream (Sparus aurata) and Sea bass (Dicentrarchus labrax) produced in two estuarine ecosystems in Andalusia (Spain): the estuary of the river Guadalquivir (La Puebla del Río, Sevilla) (A), and the estuary of the river Guadiana (Ayamonte, Huelva) (B). The collected fish individuals and water were analysed for hygiene indicator microorganisms and pathogens. The statistical analysis of results revealed that microbial counts for the different microbiological parameters were not statistically different for fish type. On the contrary, considering anatomic part, viscera showed significantly higher concentrations for Enterobacteriaceae, total coliforms and for Staphylococcus spp. coagulase +. Furthermore, location A showed in water and fish higher levels for lactic acid bacteria, aerobic mesophilic bacteria, Enterobacteriaceae, total coliforms and Staphylococcus spp. coagulase +. Neither Listeria monocytogenes, nor Salmonella spp. were detected, though Vibrio parahaemolyticus was identified, molecularly, in estuarine water in location B. The predictive analysis demonstrated that the initial microbiological quality could have an impact on product shelf-life, being longer for location B, with better microbiological quality. Results stress the relevance of preventing the microbiological contamination of water in estuary production systems in order to assure the quality and safety of Gilthead sea bream and Sea bass.