Background/Objectives: Streptococcus pneumoniae is a leading respiratory pathogen responsible for significant morbidity and mortality, particularly among vulnerable populations. Understanding its antimicrobial resistance patterns and serotype distribution is crucial for guiding treatment and prevention strategies. This study aims to examine these trends in S. pneumoniae isolates from Tunisia over a two-decade period (2000–2019). Methods: A retrospective time series analysis was conducted on data (n = 4284) gathered from eight university hospital centers across Tunisia. Antimicrobial susceptibility testing was performed according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Pneumococcal serotypes were determined for a subset of samples from 2012 to 2019 (n = 903) using multiplex PCR and latex agglutination. Results: Penicillin G resistance decreased from 9–13.7% during 2000–2002 to 4.3% by 2019, while amoxicillin resistance increased until reaching 10% in 2019. Erythromycin resistance initially increased before stabilizing between 61.9% and 66.3% during 2014–2019, whereas tetracycline resistance declined from 2000 to 2008 and fluctuated around 40% during 2009–2019. Levofloxacin resistance did not exceed 1.2% throughout the study period. The most prevalent serotypes were 14, 19F, 19A, 23F, 3, 6B, 6A, and 9V. Among them, serotype 3 was the most susceptible overall. Serotypes 23F, 14, 9V, and 6B displayed the highest levels of multi-drug resistance. Conclusions: Penicillin G (high-dosage), cefotaxime, and levofloxacin are still effective against most S. pneumoniae strains in Tunisia, while erythromycin and tetracycline are not reliable options for treating pneumococcal infections. Alarming resistance rates among prevalent serotypes, except serotype 3, underscore the need for preventive measures, rational antibiotic use, and ongoing surveillance.

The changing epidemiological profile of invasive Haemophilus influenzae infections (IIHi) is noted in the post-vaccination era. The aim of this study was to characterize phenotypically and genotypically invasive Haemophilus influenzae (Hi) isolates detected in Tunisian pediatric patients. A retrospective study was conducted in the microbiology laboratory of the Children’s Hospital of Tunis over ten years (2013–2023). All IIHi cases were included. Molecular identification and serotyping were conducted through qPCR. Molecular typing and analysis of resistance genes were extracted from whole genome sequencing data. Fifty-three IIHi cases were collected. Children under five years old were the most affected (81%). Non-typable isolates (NTHi) were predominant (79%) followed by serotype b (17%) and serotype a (4%). Genetic diversity was observed, essentially among NTHi isolates. Resistance of Hi isolates to ampicillin, amoxicillin–clavulanic acid and cefotaxime (CTX) were 42%, 20% and 4%, respectively. Thirteen isolates (29%) produced a beta-lactamase and 14 carried the blaTEM-1 gene (kappa = 0.95). For non-enzymatic resistance, group 3 (n = 12) showed resistance to ampicillin. Groupe 4 (n = 9, NTHi) showed discordances with resistance to CTX. The emergence of resistance to CTX is concerning. Continuous surveillance through molecular tools in conjunction with phenotypic and clinical data is necessary to ensure better management of these infections.

Background/Objectives: Streptococcus pneumoniae (S. pneumoniae) remains a leading cause of invasive bacterial disease in children worldwide. In Tunisia, the 10-valent pneumococcal conjugate vaccine (PCV10) was introduced into the national immunization program in 2019 for children under two years of age. This study aimed to assess molecular epidemiology, antimicrobial resistance, and vaccine impact on pediatric invasive pneumococcal disease (IPD) before and after PCV10 introduction. Methods: A retrospective study was conducted at Bechir Hamza Children’s Hospital (Tunis, Tunisia) between 2016 and 2022. IPD isolates were characterized by multiplex PCR, antimicrobial susceptibility testing, and whole-genome sequencing. Serotyping was performed using three approaches: multiplex PCR, SeroBA, and a novel cpsB gene-based algorithm. Genomic diversity and population structure were analyzed through molecular typing approaches. Incidence trends were calculated using national population data. Results: Among 150 confirmed IPD isolates, vaccine-type (VT-PCV10) strains decreased significantly from 69.8% before to 47.2% after vaccine introduction (p = 0.013), with serotype 14 showing the largest decline. Genomic analysis identified 43 sequence types and 27 global pneumococcal sequence clusters, reflecting high genetic heterogeneity. The cpsB approach demonstrated strong concordance with PCR (κ = 0.67) and SeroBA (κ = 0.85). The mean annual incidence of VT disease in children aged 0–4 years declined from 1.28 to 0.86 cases per 100,000 population, while non-vaccine serotypes showed a modest increase. Conclusions: PCV10 introduction was associated with a marked reduction in vaccine-type IPD among young children, supporting its public health benefit. Ongoing genomic surveillance remains essential to monitor serotype replacement and antimicrobial resistance in Tunisia.

Accurate identification of Mitis group streptococci especially Streptococcus pneumoniae and Streptococcus pseudopneumoniae seems difficult due to the lack of specific and sensitive tests. We performed an approach for the identification of atypical pneumococci in pediatric Tunisian population. In this study, 49 streptococcal isolates that were considered as atypical S. pneumoniae were analyzed by: optochin susceptibility in ambient and 5% CO₂ atmosphere, oxgall disk sensitivity, PCR targeting several genes and antimicrobial susceptibility. The combined results of biochemical and molecular methods showed the presence of 23 S. pneumoniae, 7 S. pseudopneumoniae, and 19 other mitis group. Among S. pseudopneumoniae, all isolates were collected from respiratory tract samples and showed a high level of resistance to β-lactams with a MIC90 of 32 mg L⁻¹. Two isolates of S. pseudopneumoniae showed the typical phenotype of optochin resistance described in the literature. All isolates could be identified only by molecular tests. Among Streptococcus pneumonaie, all strains harbored the lytA gene and the Spn9802 fragment. But only 14 strains were encapsulated. This study describes several assays for the identification of atypical pneumococci in order to gain insights on the nature of isolate and raise alert about the presence of these strains in the pediatric Tunisian community.