Ilhem Boutiba-Ben Boubaker

Influenza sentinel surveillance in Libya was formally established in 2022 by the Libyan National Center for Disease Control (NCDC). Between 2022 and 2024, a total of 1864 nasopharyngeal specimens were collected from patients presenting with influenza-like illness and tested using the GeneXpert for influenza A virus, influenza B virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and respiratory syncytial virus (RSV). Influenza A virus was detected in 21.1% (393/1864) of samples and influenza B virus was detected in 5.4% of samples (100/1864). SARS-CoV-2 and RSV were identified in 11.6% (216/1864) and 4.1% (77/1864) of specimens, respectively. A subset of 22 influenza A-positive samples was selected based on sample availability and sufficient remaining volume after the initial test for confirmatory testing and further molecular characterization. Real-time RT-PCR subtyping identified 11 A(H1N1)pdm09 and four A(H3N2) viruses. Whole-genome sequencing was successfully performed for 11 isolates, followed by phylogenetic analysis. Genetic characterization revealed that all A(H1N1)pdm09 viruses belonged to clade 6B.1A.5a.2a (5a.2a), while A(H3N2) viruses clustered within clade 3C.2a1b.2a.2a.3a.1 (2a.3a.1) were based on hemagglutinin gene mutations. No neuraminidase mutations associated with antiviral resistance were detected. This study represents the first molecular and phylogenetic characterization of circulating human influenza viruses in Libya, with sequence data submitted to the Global Initiative on Sharing All Influenza Data (GISAID) to establish baseline genetic data for influenza viruses in Libya.

Background/Objectives: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major nosocomial pathogen. Although newer agents have reduced colistin use in high-income countries, this polymyxin remains important in many low- and middle-income settings. Colistin resistance in K. pneumoniae is most commonly associated with chromosomal alterations affecting the MgrB–PhoPQ pathway, or with plasmid-mediated mcr genes. This study aimed to investigate chromosomally mediated colistin resistance in CRKP clinical isolates from a Tunisian tertiary hospital. Methods: Between 2010 and 2015, 317 non-duplicate CRKP isolates were collected at Charles Nicolle Hospital, Tunis. Colistin MICs were determined by broth microdilution. Phenotypic tests and PCR characterized carbapenemases, extended-spectrum β-lactamases, AmpC, plasmid-mediated quinolone resistance, mcr and virulence genes. Porins (OmpK35/OmpK36) and the mgrB, phoP and phoQ loci were analyzed by SDS-PAGE and sequencing. Clonal relatedness was assessed by ERIC-PCR and multilocus sequence typing. We additionally compared colistin-resistant isolates with a panel of colistin-susceptible CRKP controls and assessed phenotypic stability after serial passages without colistin. Results: Five isolates (1.6%) were colistin-resistant. All were multidrug-resistant, produced OXA-48, and two also carried NDM-1. The isolates belonged to five distinct sequence types, including high-risk clones (ST11, ST101, ST147). No mcr genes were detected. Four isolates carried disruptive mutations in mgrB, and the remaining strain harbored inactivating mutations in both phoP and phoQ with an intact mgrB. Truncating alterations in PhoP/PhoQ and frequent loss or truncation of OmpK35/OmpK36 were observed. No mgrB/phoP/phoQ alterations were detected among colistin-susceptible controls, and colistin MICs remained stable after 7 days of drug-free passaging. Conclusions: In Tunisian CRKP, colistin resistance was associated with chromosomal alterations, predominantly involving disruption of the MgrB–PhoPQ pathway, in the absence of mcr genes. These mechanisms in both high-risk and emerging sequence types underscore the adaptability of CRKP and the need for surveillance where colistin remains an important therapeutic option.

Highly mutated Omicron sub-lineages JN.1 and NB.1.8.1 harbor extensive spike changes, but their impact in preterm infants is poorly documented. We report a preterm male infant with three hospitalizations in seven weeks: severe SARS-CoV-2 ARDS at 40 days of life (DOL 40) requiring ventilation caused by JN.1.16, HCoV-OC43 infection at DOL 65, and a mild SARS-CoV-2 reinfection at DOL 87 due to NB.1.8.1, the first detection of this variant in Tunisia. Spike analysis showed a shared JN.1 backbone but distinct N-terminal and receptor-binding domain changes, supporting intra-Omicron reinfection driven by antigenic divergence and immature immunity and underscoring the value of pediatric genomic surveillance, including phylogenetic placement of case genomes within local Omicron diversity.

Background/Objectives: Multidrug-resistant (MDR) Escherichia coli resistant to third-generation cephalosporins are a growing One Health concern, but data on extraintestinal pathogenic E. coli (ExPEC) from wildlife in North Africa remain scarce. We aimed to characterize ESBL/AmpC-producing ExPEC from captive wild mammals in Tunisia and to situate these isolates in a global genomic context. Methods: In 2018, 30 fecal samples from 14 captive wild mammals in a private farm were screened on cefotaxime agar. Four cefotaxime-resistant E. coli isolates were recovered from a llama, lion, hyena, and tiger. Antimicrobial susceptibility testing and Illumina whole-genome sequencing were combined with in silico typing, resistome and virulome profiling, plasmid and mobile element analysis, human pathogenicity prediction and core-genome MLST-based minimum-spanning trees. Results: All isolates were MDR but remained susceptible to carbapenems, colistin and tigecycline. Two ST162/B1 isolates from the llama and tiger carried bla_CMY-2, whereas two ST69/D isolates from the lion and hyena harbored bla_CTX-M-15 and qnrS1. Genomes encoded 61–68 antimicrobial resistance genes and 114–131 virulence-associated genes, together with IncF-, IncI1- and IncY-type plasmids and IS26-rich insertion sequence profiles. Mating-out assays yielded cefotaxime-resistant transconjugants, supporting plasmid transferability of bla_CMY-2 or bla_CTX-M-15. PathogenFinder predicted a ≥0.93 probability of human pathogenicity for all isolates. cgMLST-based trees showed that Tunisian ST69 and ST162 clustered within internationally disseminated lineages containing human, animal and food isolates, rather than forming wildlife-restricted branches. Conclusions: Captive wild mammals in Tunisia can harbor high-risk ExPEC lineages combining ESBL/AmpC production, multidrug resistance and extensive virulence and mobility gene repertoires. These findings highlight captive wildlife as potential reservoirs and sentinels of clinically relevant E. coli and underscore the need for integrated WGS-based One Health surveillance at the human–animal–environment interface in North Africa.

Influenza sentinel surveillance in Libya was formally established in 2022 by the Libyan National Center for Disease Control (NCDC), initially comprising a single sentinel site in Tripoli. By the end of 2025, the network had expanded to 15 sites across five cities nationwide. Between 2022 and 2024, a total of 1,864 nasopharyngeal specimens were collected from patients presenting with influenza-like illness and tested using the GeneXpert for influenza A virus, influenza B virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and respiratory syncytial virus (RSV). Influenza A virus was detected in 21.1% (393/1,864) of samples and influenza B virus in 5.4% (100/1,864). SARS-CoV-2 and RSV were identified in 11.6% (216/1,864) and 4.1% (77/1,864) of specimens, respectively. A subset of 29 influenza A–positive samples was randomly selected for confirmatory testing and further molecular characterization. Real-time RT-PCR subtyping identified 13 A(H1N1)pdm09 and five A(H3N2) viruses. Whole-genome sequencing was successfully performed for 13 isolates, followed by phylogenetic analysis. Genetic characterization revealed that all A(H1N1)pdm09 viruses belonged to clade 6B.1A.5a.2a (5a.2a), while A(H3N2) viruses clustered within clade 3C.2a1b.2a.2a.3a.1 (2a.3a.1) based on hemagglutinin gene mutations. No neuraminidase mutations associated with antiviral resistance were detected. This study represents the first molecular and phylogenetic characterization of circulating human influenza viruses in Libya, with sequence data submitted to the Global Initiative on Sharing All Influenza Data (GISAID). These findings establish baseline genetic data for influenza viruses in Libya and support the strengthening of national respiratory virus surveillance.

Background/Objectives: Multidrug-resistant (MDR) Escherichia coli resistant to third-generation cephalosporins are a growing One Health concern, but data on extraintestinal pathogenic E. coli (ExPEC) from wildlife in North Africa remain scarce. We aimed to characterize ESBL/AmpC-producing ExPEC from captive wild mammals in Tunisia and to situate these isolates in a global genomic context. Methods: In 2018, 30 fecal samples from 14 captive wild mammals in a private farm were screened on cefotaxime agar. Four resistant E. coli were recovered from a llama, lion, hyena and tiger. Antimicrobial susceptibility testing and Illumina whole-genome sequencing were combined with in silico typing, resistome and virulome profiling, plasmid and mobile element analysis, human pathogenicity prediction and core-genome MLST-based minimum-spanning trees. Results: All isolates were MDR but remained susceptible to carbapenems, colistin and tigecycline. Two ST162/B1 isolates from the llama and tiger carried bla_CMY-2, whereas two ST69/D isolates from the lion and hyena harbored bla_CTX-M-15 and qnrS1. Genomes encoded 61–68 antimicrobial resistance genes and 114–131 virulence-associated genes, together with IncF-, IncI1- and IncY-type plasmids and IS26-rich insertion sequence profiles. PathogenFinder predicted a ≥0.93 probability of human pathogenicity for all isolates. cgMLST-based trees showed that Tunisian ST69 and ST162 clustered within internationally disseminated lineages containing human, animal and food isolates, rather than forming wildlife-restricted branches. Conclusions: Captive wild mammals in Tunisia can harbor high-risk ExPEC lineages combining ESBL/AmpC production, multidrug resistance and extensive virulence and mobility gene repertoires. These findings highlight captive wildlife as potential reservoirs and sentinels of clinically relevant E. coli and underscore the need for integrated WGS-based One Health surveillance at the human–animal–environment interface in North Africa.

Background/Objectives: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major nosocomial pathogen. Although newer agents have reduced colistin use in high-income countries, this polymyxin remains important in many low- and middle-income settings. Colistin resistance in K. pneumoniae is mainly driven by chromosomal changes in the MgrB–PhoPQ pathway or plasmid-mediated mcr genes. This study aimed to investigate chromosomally mediated colistin resistance in CRKP clinical isolates from a Tunisian ter-tiary hospital. Methods: Between 2010 and 2015, 317 non-duplicate CRKP isolates were collected at Charles Nicolle Hospital, Tunis. Colistin MICs were determined by broth microdilution. Phenotypic tests and PCR characterized carbapenemases, extended-spectrum β-lactamases, AmpC, plasmid-mediated quinolone resistance, mcr and virulence genes. Porins (OmpK35/OmpK36) and the mgrB, phoP and phoQ loci were analyzed by SDS-PAGE and sequencing. Clonal relatedness was assessed by ERIC-PCR and multi-locus sequence typing. Results: Five isolates (1.6%) were colistin-resistant. All were multidrug-resistant, pro-duced OXA-48 and two also carried NDM-1. The isolates belonged to five distinct sequence types, including high-risk clones (ST11, ST101, ST147). No mcr genes were detected. Four isolates carried disruptive mutations in mgrB, and the remaining strain harbored inactivating mutations in both phoP and phoQ with an intact mgrB. Truncating alterations in PhoP/PhoQ and frequent loss or truncation of OmpK35/OmpK36 were observed. Conclusions: In Tunisian CRKP, colistin resistance was mediated by chromosomal alterations, primarily disruption of the MgrB–PhoPQ pathway, in the absence of mcr genes. These mechanisms in both high-risk and emerging sequence types underscore the adaptability of CRKP and the need for surveillance where colistin remains an important therapeutic option.

BACKGROUND: Ciprofloxacin resistant Klebsiella pneumoniae is common or emerging in many geographies, and knowledge of local resistance rates is important for empirical therapy. Whilst there are known K. pneumoniae ciprofloxacin resistance determinants, there is a lack of systematic data on the effect of determinants, alone and in combination, and there are no publicly accessible tools for predicting resistance from whole genome sequence data. METHODS: The KlebNET-GSP AMR Genotype-Phenotype Group aggregated a matched genotype-phenotype dataset of n=12,167 K. pneumoniae species complex ( Kp SC) isolates from 27 countries between 2001-2021. We developed a rules-based classifier to predict ciprofloxacin resistance by categorizing the number of quinolone resistance determining regions mutations in gyrA and parC , the number of plasmid-mediated quinolone resistance genes, and the presence/absence of aac(6ʹ)-Ib-cr (which can acetylate ciprofloxacin). Predictive performance was assessed using the discovery dataset, for which we re-phenotyped discrepant isolates; and validated using externally contributed datasets (n=7,030 Kp SC isolates). RESULTS: The rules-based classifier predicted R vs S/I with categorical agreement, sensitivity, and specificity >96%, and major/very major error rates Kp SC sources (human, animal, other), species, and intra-species lineages. External validation of the classifier yielded overall 93.12% categorical agreement [95% confidence interval (CI), 92.50-93.74%], 8.65% major errors [95% CI, 7.34-9.97%], and 6.20% very major errors [95% CI, 5.51-6.90%]. We implemented the classifier in Kleborate, a command-line tool that is integrated into the Pathogenwatch web platform. Using this to assess the global distribution of ciprofloxacin resistance determinants in Kp SC genomes available in Pathogenwatch (n=31,319, from 109 countries between years 2000-2023), we observed a significant positive association between national quinolone consumption rates and predicted ciprofloxacin resistance (R ² =0.20, p=0.004). CONCLUSIONS Ciprofloxacin resistance phenotypes can be reasonably predicted from genotypes, which is sufficient for informing surveillance. However, unexplained resistance remains and accuracy is insufficient for clinical applications. We demonstrate the value of aggregating genotype-phenotype data to explore resistance mechanisms and develop predictors, but highlight complexities in combining phenotype data from different assays and standards.

Background/Objectives: As antimicrobial resistance patterns of Gram-negative bacteria change over time, this study aimed to analyze the antimicrobial susceptibility trends of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii isolates in Tunisia. Methods: From 1999 to 2019, non-duplicate isolates of Gram-negative bacteria were collected from 11 Tunisian hospitals as part of an antimicrobial resistance surveillance program. Antimicrobial susceptibility testing was performed using the disk diffusion method according to the European Committee on Antimicrobial Susceptibility Testing guidelines. Results: Out of 213,434 isolates collected during the study period, 58.8% were E. coli, 22% were K. pneumoniae, 14.4% were A. baumannii, and 4.8% were P. aeruginosa, with 67% of the isolates sourced from urine samples. E. coli showed a significant increase in resistance to third-generation cephalosporins (3GC), from 5.4% in 2004 to 16.5% in 2019, but K. pneumoniae displayed a rising trend of resistance to imipenem, from 1% in 2005 to 18.6% in 2019; meanwhile, amikacin remained effective against K. pneumoniae isolates. P. aeruginosa did not exhibit a significant change in resistance to imipenem. A. baumannii had a high resistance rate to imipenem that increased from 34.5% in 2008 to 84.2% in 2019 and had low susceptibility rates to all other antibiotics tested. Conclusions: This study reveals high carbapenem resistance among K. pneumoniae and A. baumannii in Tunisia. A. baumannii shows alarming multidrug resistance that requires urgent control measures.

The antimicrobial resistance (AMR) surveillance network has been monitoring bloodstream bacterial pathogens and their resistance since 1999 in Tunisia. We report the long-term trends in the distribution of bloodstream bacterial pathogens and their resistance patterns from this surveillance database. We analyzed antibiotic resistance rates in 11 tertiary teaching hospital laboratories under the AMR surveillance network during 2011–2023, focusing on six priority bacterial pathogens, using the Cochrane–Armitage test for trend analysis. Of 22,795 isolates, K. pneumoniae (38.5%) was the most common, followed by S. aureus (20.4%), E. coli (13.6%), and A. baumannii (10.3%). Carbapenem resistance was highest in A. baumannii (77%), followed by Pseudomonas aeruginosa (29.3%), K. pneumoniae (19.4%), and Enterobacter cloacae (6.8%). Carbapenem-resistant Enterobacterales and third-generation cephalosporin-resistant Enterobacterales (3GCREB) increased from 10.6% to 26.3% (p-value < 0.001), and from 39% to 50.2%, respectively, during 2011–2023 (p-value < 0.001). Vancomycin resistance (38.3%) and the emergence of linezolid resistance in 2019 (2.4%) were reported in E. faecium isolates. Resistance to carbapenems and 3GC is a major challenge to controlling BSI in Tunisia. The national AMR surveillance network helps monitor annual patterns and guides empirical therapy. An integrated database combining clinical profiles and resistance data via real-time data-sharing platforms could improve clinical decision-making.