Pseudomonas aeruginosa is a major cause of health care-associated infections, particularly in intensive care units (ICUs). To the best of our knowledge, this is one of the rare studies reporting the prevalence and antimicrobial resistance patterns of P. aeruginosa in a Tunisian ICU. This retrospective study included all P. aeruginosa strains isolated from ICU patients in Sfax, Tunisia, between 2017 and 2024. A total of 1,126 nonredundant P. aeruginosa strains were obtained from 767 patients. P. aeruginosa strains accounted for 21.1% of all clinical isolates in the ICU. The majority of strains were isolated from respiratory samples (54.6%) and blood cultures (14.3%). The prevalence of resistance rates was highest for ticarcillin (48.3%) and ticarcillin-clavulanic acid (48.6%). The resistance to both ceftazidime and imipenem was observed in 33.6% of P. aeruginosa strains. The prevalence of resistance of P. aeruginosa was 29.8% and 26.2% to ceftazidime-avibactam (CAV) and ceftolozane-tazobactam (C/T), respectively. The prevalence of resistance to amikacin and ciprofloxacin was 36.2% and 37.9%, respectively. Of the 1,126 isolates, 346 (30.7%) were multidrug-resistant P. aeruginosa (MDR-PA) strains, presenting resistance to ceftazidime, ciprofloxacin, and amikacin simultaneously. Since 2022, a significant increase in the MDR rate was observed, reaching 46.3% in 2024 (P <0.001). The resistance rates of MDR-PA were 52.5% and 56.8% to C/T and CAV, respectively. None of the tested MDR-PA strains showed resistance to colistin. This study highlights a high prevalence of MDR strains of P. aeruginosa in our ICU, with increasing resistance over recent years. Prevention is highly warranted.

Purpose. Invasive pneumococcal disease (IPD) remains a major global public health concern due to its high morbidity and mortality rates, particularly among children and the elderly. This study aimed to apply whole-genome sequencing (WGS) to characterize Streptococcus pneumoniae strains responsible for IPD in south Tunisia, including serotype distribution, clonal relationship and antimicrobial resistance (AMR) profiles. Methods. A total of 148 IPD S. pneumoniae isolates were collected from the microbiology laboratory at Habib Bourguiba University Hospital in Sfax, Tunisia, between 2012 and 2022. These isolates underwent WGS using Illumina technology. Bioinformatic analyses were performed to determine serotype distribution, sequence types (STs), Global Pneumococcal Sequence Clusters (GPSCs), phylogenetic relationships and AMR determinants. Results. Twenty-six different serotypes were identified, with the most prevalent being 14 (18%), 3 (13%), 19A (12%) and 19F (11%). The isolates showed high genomic diversity, as they belonged to 32 GPSCs and 59 STs. The most common GPSCs were GPSC-6, GPSC-10 and GPSC-44, associated with serotypes 14, 19A and 19F, respectively. The most frequent STs were ST2918, ST179 and ST3772. The most common resistance genes were erm B (53%) and tet M (55%), which were linked to resistance against erythromycin and tetracycline, respectively. There was a considerable concordance between WGS-based and phenotypic resistance profiles for most tested antibiotics, with few major and very major errors for most antibiotics. Temporal analysis showed a decline in serotypes 19F and 9V throughout the study period, which was associated with slight decreases in GPSC-6 and GPSC-44, while serotype 19A and GPSC-10 sharply increased. Conclusion. This study highlights the substantial genomic diversity, serotype distribution and high prevalence of AMR among IPD S. pneumoniae isolates in south Tunisia, underscoring the need for continued surveillance and effective vaccination strategies to combat this persistent public health threat.

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.

Peritonitis is one of the most serious complications of peritoneal dialysis (PD). The most common microbial causes of PD-associated peritonitis are Gram-positive pathogens. However, unusual microorganisms may be involved in this type of infection, such as Campylobacter fetus. Here, we report a case of C. fetus peritonitis in a 60-year-old woman undergoing continuous ambulatory PD. This patient was admitted to the nephrology department with a history of fatigue, abdominal pain, diarrhea, vomiting, and fever. A bacteriological examination of the PD effluent revealed a cloudy liquid containing 900 leukocytes/mm3 with 75% polymorphonuclear neutrophils. C. fetus was obtained in a pure culture. Antibiotic susceptibility testing was performed. A favorable outcome was noted after treatment with amoxicillin or clavulanic acid and amikacin. Thus, an accurate bacteriological diagnosis of PD-associated peritonitis requires optimal culture methods to improve the isolation of fastidious microorganisms such as Campylobacter and therefore guide appropriate antibiotic therapy.

Brucella melitensis is a zoonotic pathogen that poses a worldwide public health challenge. In recent years, whole-genome sequencing has become a widely accepted molecular typing method for the genomic epidemiology of brucellosis. This study reports the genomic characteristics of 24 B. melitensis strains isolated from human infections in southern Tunisia over 35 years (1988–2022). We utilized WGS to analyze the clonal relationships of these strains, their relatedness to international sequences, their antimicrobial resistance determinants, and their virulence factors. Our findings revealed a high genetic stability over three decades. All isolates were identified as B. melitensis biovar 3 and were assigned to the same sequence type, ST11, using the MLST-9 scheme. Using the MLST-21, Tunisian sequences shared 20 out of 21 alleles and were assigned to 2 closely related STs (ST89 and ST114). Phylogenetic analysis indicated that all Tunisian sequences were grouped into a single subcluster within lineage I, the West Mediterranean clade, and were highly related to other strains from the Maghreb region (Morocco and Algeria). Antimicrobial resistance analysis revealed no classical resistance determinants. However, mprF, bepCDEFG genes, and missense mutations in rpoB, gyrA, gyrB, and parC genes were identified. Virulence analysis identified 67 genes, predominantly involved in lipopolysaccharide biosynthesis and the type IV secretion system. To our knowledge, this study represents the first genomic investigation of B. melitensis strains circulating in Tunisia. Our findings underscore the importance of genomic surveillance in understanding the epidemiology and evolution of brucellosis in North Africa.

Purpose. The prevalence of multidrug-resistant (MDR) Shigella sonnei is increasing globally, raising concerns for public health. In 2022, an outbreak of MDR S. sonnei was observed in Tunisia. We aimed to evaluate the genetic profile of S. sonnei isolates during the outbreak, including their clonal relationship, antimicrobial determinants and connection to international strains.Methods. In this study, we sequenced the whole genome of 24 S. sonnei strains collected from South Tunisia between July 2022 and November 2023. Bioinformatic analysis was conducted to confirm species identification, assign sequence types, determine core genome sequence types, analyse phylogenetic relationships and identify antimicrobial resistance determinants. Phylodynamic and phylogeographic analyses were performed to trace the spatiotemporal spread of the outbreak genotype.Results. Our investigation revealed that 23 out of 24 isolates were grouped into the HC10-20662 genotype within the 3.6.3 subclade. All isolates carried the blaCTX-M-15 gene associated with extended-spectrum beta-lactamase production, as well as the dfrA1 and qnrS1 genes, along with the D87G mutation in gyrA. Additionally, the sul2, tet(A) and mph(A) resistance genes were present in most isolates (96%, 96 and 83, respectively). Phylogeographic analysis suggested that the outbreak genotype likely spread in Europe before being introduced into Tunisia.Conclusion. To the best of our knowledge, this is the first MDR S. sonnei outbreak in the country. The HC10-20662 genotype appears to be responsible for a multi-country outbreak, affecting both Tunisia and Europe. Continued genomic surveillance efforts, both nationally and internationally, are essential for monitoring the dynamic evolution and global spread of MDR S. sonnei.

INTRODUCTION:In the lack of updated Tunisian epidemiological data, we sought to describe the epidemiology of Group B Streptococcus (GBS) in pregnant women and newborns.MATERIALS AND METHODS:A retrospective analysis of GBS neonatal invasive infections and a cross-sectional study evaluating the prevalence of maternal GBS colonization were conducted. GBS isolates were tested for antimicrobial susceptibility, serotyped, and assessed for the appurtenance to the hypervirulent ST17 clone.RESULTS:Of 98 neonates with GBS, early-onset GBS disease (EOD) comprised 83.7 and 16.3% were late-onset GBS disease (LOD). The prevalence of maternal GBS colonization was 27%. All GBS isolates were susceptible to penicillin. Serotype III predominated (42.6%) for neonatal invasive infections. GBS isolates belonging to the ST17 sequence type were found only as serotype III.CONCLUSION:This study documents the frequency of GBS EOD, the high rate of maternal GBS colonization, and the predominance of the hypervirulent clone type III/ST17 in infants.

We aimed to assess the prevalence of nasopharyngeal pneumococcal carriage and to determine serotype distribution, antibiotic susceptibility patterns, and evolutionary dynamics of Streptococcus pneumoniae isolates in healthy under-five children. Nasopharyngeal swabs were collected from healthy children over three survey periods between 2020 and 2022. All pneumococcal isolates were serotyped and tested for antimicrobial susceptibility. A total of 309 S. pneumoniae isolates were collected, with an overall prevalence of nasopharyngeal pneumococcal carriage of 24.4% (CI95%: [22–26.8%]). These isolates were classified into 25 different serotypes. The most common serotypes were 14 (14.9%), 19F (12%), 6B (10.4%), and 23F (7.4%), which are covered by the PCV10 vaccine, as well as 19A (8.4%) and 6A (7.8%), which are covered by the PCV13 vaccine. A significant decrease in the proportion of serotype 19F (p = 0.001) and an increase in serotypes 19A (p = 0.034) and 6A (p = 0.029) were observed between the three survey periods. Multidrug resistance (MDR) was noted for 56.6% of the isolates. A significant association with antimicrobial resistance was observed for the most frequent serotypes, mainly serotype 19A. In conclusion, one-quarter of healthy under-five children in Tunisia carried S. pneumoniae in their nasopharynx. A dominance of vaccine serotypes significantly associated with antimicrobial resistance was recorded.

Parachlamydia acanthamoebae and Simkania negevensis, two Chlamydia-like bacteria, have been recently recognized as emerging human respiratory pathogens. The prevalence and frequency of these bacteria in the environment and among atypical pneumonia patients are still underestimated by classical cultures, immunohistochemistry and serology which are non-specific, long and tedious methods. This study aims to develop a new duplex probe-based q-PCR assay for the simultaneous detection and quantification of P. acanthamoebae and S. negevensis. The selected hydrolysis probes displayed no cross-reaction with the closely related Chlamydia or the other tested waterborne pathogens. The assay achieved a large dynamic range for quantification (from 5 × 106 to 5 DNA copies/reaction). Efficiencies of FAM and JOE label probes weren't affected when they were combined. They were close to 100%, indicating the linear amplification. The application of this diagnostic tool resulted in 9/47 (19%) and 4/47 (8.5%) positive water samples for P. acanthamoebae and S. negevensis, respectively. P. acanthamoebae was also covered from 2/78 (2.5%) respiratory specimens and only one case (1/200 = 0.5%) of P. acanthamoebae and SARS-CoV-2 co-infection was noticed. While S. negevensis wasn't detected in clinical samples, the developed duplex q-PCR was shown to be an accurate, highly sensitive, and robust diagnostic tool for the detection and quantification of P. acanthamoebae and S. negevensis.

Abstract Background Streptococcus pneumoniae remains a leading cause of morbidity and mortality worldwide. In this study, we sought to analyze serotype distributions, antibiotic resistance, and genetic relationships of 106 clinical invasive pneumococcal isolates recovered in Tunisia between 2012 and 2018, prior to the routine use of pneumococcal conjugate vaccines (PCV). Methods We used multiplex PCR, the disk diffusion method and/or E-test, and multi-locus sequence typing (MLST). Results The most frequent serotypes were 14 (17%), 19F (14.2%), and 3 (11.3%). Of the 106 S. pneumoniae isolates, 67.9% were penicillin non-susceptible (29.4% were resistant), 45.3% were amoxicillin non-susceptible (17% were resistant), and 16% were cefotaxime non-susceptible. For antibiotics other than β-lactams, resistance rates to erythromycin, tetracycline, cotrimoxazole, and chloramphenicol were 62.3, 33, 22.6, and 4.7%, respectively. Two isolates were non-susceptible to levofloxacin. Among 66 erythromycin-resistant pneumococci, 77.3% exhibited the cMLSB phenotype, and 87.9% carried ermB gene. All tetracycline-resistant strains harbored the tetM gene. The potential coverage by 7-, 10-, and 13-valent pneumococcal conjugate vaccines were 55.7, 57.5, and 81.1%, respectively. A multilocus sequence typing analysis revealed great diversity. Fifty different sequence types (STs) were identified. These STs were assigned to 10 clonal complexes and 32 singletons. The most common STs were 179, 2918, 386, and 3772 – related mainly to 19F, 14, 6B/C, and 19A serotypes, respectively. Conclusions This study demonstrated that the majority of the serotypes of invasive pneumococci in the Tunisian population were 14, 19F, and 3. Moreover, we noted a high degree of genetic diversity among invasive S. pneumoniae isolates. The highest proportions of antibiotic non-susceptible isolates were for penicillin, erythromycin, and tetracycline. Further molecular characteristics are required to monitor the genetic variations and to follow the emergence of resistant pneumococci for the post-vaccination era in Tunisia.

Human respiratory infections caused by a large variety of microbial pathogens are the most common diseases responsible for hospitalization, morbidity and mortality. Parachlamydia acanthamoebae , a Chlamydia -related bacterium, has been found to be potentially associated with these diseases. An early and accurate diagnosis of this pathogen could be useful to avoid the potential respiratory complications linked especially to COVID-19 patients and to set suitable outbreak control measures. A TaqMan-PCR assay was developed to detect and quantify Parachlamydia acanthamoebae in environmental and clinical samples from patients of all ages with COVID-19. The selected hydrolysis probe displayed no cross-reaction with the closely related Chlamydia or the other tested pathogens. This q-PCR achieved good reproducibility and repeatability with a detection limit of about 5 DNA copies per reaction. Using this q-PCR assay, Parachlamydia acanthamoebae was detected in 2/78 respiratory specimens and 9/47 water samples. Only one case (1.3%) of Parachlamydia acanthamoebae and SARS-COV-2 co-infection was noticed. To our knowledge, the combination of these two respiratory pathogens has not been described yet. This new TaqMan-PCR assay represents an efficient diagnostic tool to survey Parachlamydia acanthamoebae on a large-scale screening programs and also during outbreaks.

Streptococcus pneumoniae remains a significant cause of morbidity and mortality worldwide despite the overall success of the vaccine programs. In Tunisia, pneumococcal conjugate vaccines (PCV)10 was introduced in the national immunization program in April 2019. We sought to determine the relationship between serotypes and antimicrobial nonsusceptibility of S. pneumoniae isolates recovered from clinical samples in the prevaccination period in the south of Tunisia. A total of 504 nonduplicate S. pneumoniae isolates collected between 2012 and 2018 were tested for antimicrobial susceptibility, among them 439 (87.1%) were serotyped. The most common serotypes were 19F (17.8%), 14 (15.3%), 3 (9.1%), 19A (8.2%), and 23F (7.3%). The proportions of isolates with serotypes covered by PCV7, PCV10, and PCV13 were 55.4%, 56.3%, and 77.9%, respectively. Three-quarters (74.4%) of pneumococcal isolates were nonsusceptible to penicillin, and about half (54.8%) were multidrug resistant. Penicillin nonsusceptibility was observed for all 19A and 23F isolates, and was significantly associated with serotypes 19F (odds ratio [OR]: 33.7) and 14 (OR: 8.7). A significant association with multidrug resistance was noted for serotypes 19A (OR: 10), 19F (OR: 9.4), 23F (OR: 8.6), and 6B (OR: 5.2). The alarming rates of pneumococcal antimicrobial nonsusceptibility and the strong association with the most prevalent serotypes compel microbiologists to monitor the impact of the PCV10 introduced recently in our national immunization program.

Simkania negevensis is an emerging Chlamydia-like bacterium related to human respiratory diseases. An early and accurate detection of this pathogen could be useful to monitor the potential infectious risks and to set suitable outbreak control measures. In Tunisia, distribution and abundance of S. negevensis remain until now largely unknown. In the present work, a qPCR assay, targeting the 16S rRNA gene, for fast detection and quantification of S. negevensis was developed and validated. A high specificity for S. negevensis detection displaying no cross-reaction with the closely related Chlamydia spp. or the other tested microorganisms was noticed. qPCR assay performance was considered very satisfying with detection limits of 5 DNA copies per reaction. qPCR assay validation was performed by screening 37 clinical specimens and 35 water samples. S. negevensis wasn't detected in respiratory samples, but it was found in four cases of water samples. We suggest that the qPCR assay developed in this study could be considered sufficiently characterized to initiate the quantification of S. negevensis in environmental samples.