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The widespread use of pesticides, particularly in combinations, has resulted in enhanced hazardous health effects. However, little is known about their molecular mechanism of interactions. The aim of this study was to assess the neurotoxicity effect of pesticides in mixtures by adopting a 3D in vitro developed neurospheroid model, followed by treatment by increased concentrations of pesticides for 24 h and analysis by a shotgun proteomic-based approach with high-resolution tandem mass spectrometry. Three proteins, namely, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), α-enolase, and phosphoglycerate-kinase-1, were selected as key targets in the metabolic process. Only high doses of pesticides mitigated cell-density proliferation with the occurrence of apoptotic cells, which unlikely makes any neurological alterations in environmental regulatory exposures. The proteomic analysis showed that majority of altered proteins were implicated in cell metabolism. De novo peptide sequencing revealed ion losses and adduct formation, namely, a trityl-post-translational modification in the active site of 201-GAPDH protein. The study also highlights the plausible role of pyrethroids to be implicated in the deleterious effects of pesticides in a mixture. To the best of our knowledge, our finding is the first in toxicoproteomics to deeply elucidate pesticides' molecular interactions and their ability to adduct proteins as a pivotal role in the neurotoxicity mechanism.

The prevalence of infections caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) and carbapenemase-producing E. coli (CP-EC) is increasing worldwide. We investigated the epidemiology of ESBL-EC and CP-EC causing hospital-acquired (HA) infections in a large teaching hospital in Tunisia over the last two decades and compared it with a collection of 107 community-acquired (CA) ESBL-EC isolates. Between 2001 and 2019, the incidence of HA ESBL-EC increased significantly from 0.08 to 0.32 cases per 1,000 patient days, due entirely to the rapid emergence and expansion of ST131, which accounted for 42.3% (157/371) of HA ESBL-EC. Most ESBL-EC harbored the CTX-M type (92%) with a predominance of blaCTX-M-15. The C2/H30-Rx subclone ( n = 103, 65.6%) accounted for 90% of ST131 isolates between 2003 and 2012 and was exclusively associated with CTX-M-15, whereas cluster C1-M27, which was associated with CTX-M-27, emerged in 2013 and expanded gradually to 55% of ST131 in 2019. ST131 prevalence was higher among CA ESBL-EC than HA ESBL-EC (63.6% vs. 42.3%, P = 0.002). CA C2 subclone and non-ST131 isolates showed higher virulence scores than HA isolates. The incidence of CP-EC remained stable over the study period with a mean of 0.08 cases per 1,000 patient days. Among the 38 identified CP-EC isolates, only 16.2% belonged to the ST131 clone and 81.5% produced OXA-48-like carbapenemases. ST131 is the major driver of ESBL-EC spread in both hospital and community settings in Tunisia, mainly linked to the expansion of the CTX-M-15-C2 and CTX-M-27-C1 subclades. The emergence of CP-EC requires ongoing genomic surveillance., Importance: We aimed to investigate the microbiological features of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) and carbapenemase-producing E. coli (CP-EC) causing hospital- and community-acquired infections in Tunisia over the last two decades. The study captured the emergence and expansion of the CTX-M-15-C2 ST131 subclade and successively the CTX-M-27-C1 ST131 subclade, which were responsible for the steady increase in the prevalence of ESBL-EC. However, the incidence of CP-EC remained stable over the study period with a highly diverse content in carbapenemase genes dominated by blaOXA-48-like. This is the first study to provide comprehensive data on the epidemiology of ESBL-EC and CP-EC in a North African country.

Aim: Histology is the most widely used test to detect H. pylori . PCR is less used but allows the detection of both infection and antibiotics' resistance. Methods: We conducted a monocentric cross-sectional study, collecting 97 symptomatic patients to assess the diagnostic performance of histology in the detection of H. pylori infection compared with PCR. Results: Sensitivity of histology in comparison with PCR was 81.5% and specificity was 56.3%. A history of anti- H. pylori therapy intake, as well as the density of the bacterium on the gastric sample and the presence of gastric atrophy, were significantly correlated to the PCR's result in terms of H. pylori detection. Conclusion: Thus, histology can be considered as an efficient test compared with PCR in H. pylori detection., Competing Interests: The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties., (© 2024 Manel Moalla.)

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.

Pesticides are increasingly used in combinations in crop protection, resulting in enhanced toxicities for various organisms. Although protein adductomics is challenging, it remains a powerful bioanalytical tool to check environmental exposure and characterize xenobiotic adducts as putative toxicity biomarkers with high accuracy, facilitated by recent advances in proteomic methodologies and a mass spectrometry high-throughput technique. The present study aims to predict the potential neurotoxicity effect of imidacloprid and λ-cyhalothrin insecticides on human neural cells. Our protocol consisted first of 3D in vitro developing neurospheroids derived from human brain tumors and then treatment by pesticide mixture. Furthermore, we adopted a bottom-up proteomic-based approach using nanoflow ultraperformance liquid chromatography coupled with a high-resolution mass spectrometer for protein-adduct analysis with prediction of altered sites. Two proteins were selected, namely, calcium-calmodulin-dependent protein kinase-II (CaMK2) and annexin-A1 (ANXA1), as key targets endowed with primordial roles. De novo sequencing revealed several adduct formations in the active site of 82-ANXA1 and 228-CaMK2 as a result of neurotoxicity, predicted by the added mass shifts for the structure of electrophilic precursors. To the best of our knowledge, our study is the first to adopt a proteomic-based approach to investigate in depth pesticide molecular interactions and their potential to adduct proteins which play a crucial role in the neurotoxicity mechanism.

High-grade gliomas represent the most common group of infiltrative primary brain tumors in adults associated with high invasiveness, agressivity, and resistance to therapy, which highlights the need to develop potent drugs with novel mechanisms of action. The aim of this study is to reveal changes in proteome profiles under stressful conditions to identify prognostic biomarkers and altered apoptogenic pathways involved in the anticancer action of human isocitrate dehydrogenase (IDH) mutant high-grade gliomas. Our protocol consists first of a 3D in vitro developing neurospheroid model and then treatment by a pesticide mixture at relevant concentrations. Furthermore, we adopted an untargeted proteomic-based approach with high-resolution mass spectrometry for a comparative analysis of the differentially expressed proteins between treated and nontreated spheroids. Our analysis revealed that the majority of altered proteins were key members in glioma pathogenesis, implicated in the cellular metabolism, biological regulation, binding, and catalytic and structural activity and linked to many cascading regulatory pathways. Our finding revealed that grade-IV astrocytomas promote the downstream of the mitogen-activated-protein-kinases/extracellular-signal-regulated kinase (MAPK1/ERK2) pathway involving massive calcium influx. The gonadotrophin-releasing-hormone signaling enhances MAKP activity and may serve as a negative feedback compensating regulator. Thus, our study can pave the way for effective new therapeutic and diagnostic strategies to improve the overall survival.

Pesticides are widely used, resulting in continuing human exposure with potential health impacts. Some exposures related to agricultural works have been associated with neurological disorders. Since the 2000s, the hypothesis of the role of pesticides in the occurrence of central nervous system (CNS) tumors has been better documented in the literature. However, the etiology of childhood brain cancers still remains largely unknown. The major objective of this work was to assess the potential role of pesticide exposure as a risk factor for CNS tumors based on questionnaires and statistical analysis of information collected from patients hospitalized in the Neurosurgery Department of the Habib Bourguiba Hospital Medium in Sfax, Tunisia, during the period from January 1, 2022, to May 31, 2023. It also aimed to develop a simple and rapid analytical method by the gas chromatography-mass spectrometry technique for the research traces of pesticide metabolites in some collected human brain tumor tissues in order to more emphasize our hypothesis for such a correlation between pesticide exposure and brain tumor development. Patients with a history of high-risk exposure were selected to conduct further analysis. Chemometric methods were adapted to discern intrinsic variation between pathological and control groups and ascertain effective separation with the identification of differentially expressed metabolites accountable for such variations. Three samples revealed traces of pesticide metabolites that were mostly detected at an early age. The histopathological diagnosis was medulloblastoma for a 10-year-old child and high-grade gliomas for 27- and 35-year-old adults. The bivariate analyses (odds ratio >1 and P value <5%) confirmed the great probability of developing cancer by an exposure case. The Cox proportional hazards model revealed the risk of carcinogenicity beyond the age of 50 as a long-term effect of pesticide toxicity. Our study supports the correlation between pesticide exposure and the risk of development of human brain tumors, suggesting that preconception pesticide exposure, and possibly exposure during pregnancy, is associated with an increased childhood brain tumor risk. This hypothesis was enhanced in identifying traces of metabolites from the carbamate insecticide class known for their neurotoxicity and others from pyridazinone, organochlorines (OCs), triazole fungicide, and N-nitroso compounds known for their carcinogenicity. The 2D-OXYBLOT analysis confirmed the neurotoxicity effect of insecticides to induce oxidative damage in CNS cells. Aldicarb was implicated in brain carcinogenicity confirmed by the identification of oxime metabolites in a stress degradation study. Revealing "aziridine" metabolites from the OC class may better emphasize the theory of detecting traces of pesticide metabolites at an early age. Overall, our findings lead to the recommendation of limiting the residential use of pesticides and the support of public health policies serving this objective that we need to be vigilant in the postmarketing surveillance of human health impacts., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

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., (© 2023. The Author(s).)

Background. Helicobacter pylori (H. pylori) resistance to clarithromycin is increasing worldwide. Data on the prevalence of H. pylori resistance are limited in Tunisia. Gap statement. Given that H. pylori resistance to clarithromycin has not been studied in Tunisia since 2010, there was a need to determinate its prevalence and the principal mutations implicated in this resistance. Aim. The aims were to define the prevalence of H. pylori infection among symptomatic patients and to determinate the level of clarithromycin resistance among these patients and the main mutations conferring this resistance. Methods. We conducted a cross-sectional study from March 2017 to February 2020 in the Hepato-Gastroenterology Department of Hedi Chaker University Hospital in Sfax that included 124 Tunisian patients who underwent gastroduodenal endoscopy with biopsies. Mutations conferring resistance to clarithromycin were detected using the Allplex H. pylori and ClariR PCR Assay. Results. Out of 124 biopsies, 101 (81.5 2 %) were PCR-positive for H. pylori . Mutations conferring resistance to clarithromycin were detected in 30/95 (31.6 %) of patients. The rate of primary resistance was 25.3 % and of secondary resistance 62.5 %. The most frequently detected mutation was A2143G (86, 90%) followed by A2142G (11, 36%). Seven patients had a double mutation A2143G-A2142G. The factors independently associated with resistance to clarithromycin were diabetes, high blood pressure, the presence of a bulbar ulcer on endoscopy and the presence of gastric atrophy on histology. Conclusion. Detection of more than 25 % of strains with clarithromycin resistance mutations makes the H. pylori first-line treatment with clarithromycin questionable in our setting, and a review of empirical treatment of H. pylori is urgently needed.

Objectives: To determine the prevalence of community intestinal carriage of ESBL-producing Enterobacterales (ESBL-E), acquired-AmpC-producing Enterobacterales (aAmpC-E) and carbapenemase-producing Enterobacterales (CPE) in Tunisia., Methods: From November 2012 to September 2017, stool samples from food handlers in Sfax, Tunisia, were screened for ESBL-E, AmpC-E and CPE using antibiotic-containing media. The genes encoding these β-lactamases were characterized by PCR, sequencing, and transfer assays. ST131 clonal groups were detected by PCR and characterized for antibiotic resistance, virulence genes and PFGE patterns., Results: Of 2135 participants, ESBL-E, aAmpC-E, and CPE carriage were detected in 419 (19.63%), 35 (1.63%) and 7 (0.33%) participants, respectively. CTX-M-15 (60%), CTX-M-1 (16.8%) and CTX-M-27 (12.8%) were the most common ESBL determinants. The ESBL-E carriage was significantly higher in summer (33%) and autumn (25.7%) than in winter (12.1%) and spring (11.4%). ST131 was detected in 50 (13.2%) of the 378 ESBL-producing Escherichia coli isolates; most of them (35; 70%) belonged to subclade C1 (cluster C1-M27: 23 isolates, 46%; cluster C1-non-M27: 12 isolates, 24%) followed by those belonging to subclade C2 (11; 22%). Although subclade C2 isolates, all harbouring blaCTX-M-15, had the highest resistance rates and virulence factor and addiction system scores, the subclade C1 isolates, mainly harbouring blaCTX-M-27 (94%), were predominant since 2015. The most frequently detected carbapenemase-encoding gene was blaOXA-48-like (85%) and acquired AmpC-encoding genes were blaDHA-1 (54%) and blaCMY-2 (46%)., Conclusions: This is the first large Tunisian study to reveal a high faecal ESBL carriage rate, a low CPE carriage rate, and the predominance of CTX-M-27-producing subclade C1 among faecal ESBL-ST131 isolates in the Tunisian community., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Salmonella Enteritidis causes a major public health problem in the world. Whole genome sequencing can give us a lot of information not only about the phylogenetic relatedness of these bacteria but also in antimicrobial resistance and virulence gene predictions. In this study, we analyzed the whole genome data of 45 S. Enteritidis isolates recovered in Tunisia from different origins, human, animal, and foodborne samples. Two major lineages (A and B) were detected based on 802 SNPs differences. Among these SNPs, 493 missense SNPs were identified. A total of 349 orthologue genes mutated by one or two missense SNPs were classified in 22 functional groups with the prevalence of carbohydrate transport and metabolism group. A good correlation between genotypic antibiotic resistance profiles and phenotypic analysis were observed. Only resistant isolates carried the respective molecular resistant determinants. The investigation of virulence markers showed the distribution of 11 Salmonella pathogenicity islands (SPI) out of 23 previously described. The SPI-1 and SPI-2 genes encoding type III secretion systems were highly conserved in all isolates except one. In addition, the virulence plasmid genes were present in all isolates except two. We showed the presence of two fimbrial operons sef and ste previously considered to be specific for typhoidal Salmonella . Our collection of S. Enteritidis reveal a diversity among prophage profiles. SNPs analysis showed that missense mutations identified in fimbriae and in SPI-1 and SPI-2 genes were mostly detected in lineage B. In conclusion, WGS is a powerful application to study functional genomic determinants of S. Enteritidis such as antimicrobial resistance genes, virulence markers and prophage sequences. Further studies are needed to predict the impact of the missenses SNPs that can affect the protein functions associated with pathogenicity., Competing Interests: The authors declare that they have no competing interests., (© 2022 The Authors. Published by Elsevier B.V.)

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.

Introduction: Streptococcus pneumoniae can be responsible for severe human infections. Optochin resistance has been a potential cause of misidentification of pneumococcus and other members of the mitis group. Hence, rapid and easy optochin resistant (Optr) S. pneumoniae identification is essential., Methodology: Atypical pneumococci were characterized using optochin susceptibility, bile solubility based on spectrophotometric reading, serotyping, pulsed field gel electrophoresis (PFGE), 16S rRNA sequencing and PCR-based assays targeting pneumococcal genes lytA, ply, pspA, cpsA, Spn9802 and Spn9828., Results: Optical density values for the bile solubility test suggest the identification of four Optr S. pneumoniae and one Streptococcus pseudopneumoniae. All Optr pneumococci harbored cpsA, lytA, ply, Spn9802, Spn9828 and pspA genes. Only ply, spn9802 and Spn9828 genes were detected in S. pseudopneumoniae. The 16S rRNA sequencing differentiates between these two species. Optr S. pneumoniae strains belonged to different genotypes and serotypes (14, 19A, 3 and 9V). Three Optr S. pneumoniae isolates were typed as pspA family 2, while one belonged to pspA family 1. Sequencing of the atpA and atpC gene of the Optr variants revealed three mutations in the ATPase a-subunit (L99I, M23V and V52I) and one mutation in ATPase c-subunit (V48I)., Conclusions: Our data indicate that bile OD-values provides an accurate, fast and easy method to discriminate between Optr S. pneumoniae and other Streptococcus mitis group. Moreover molecular techniques, confirming the bile test, can be used in order to prevent these atypical pneumococci and alert clinical microbiologists of the presence of these strains in the community., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2021 Sonia Ktari, Nour El Houda Ben Ayed, Sonda Maalej, Basma Mnif, Faouzia Rhimi, Adnene Hammami.)

The treatment of acute osteomyelitis is becoming more challenging since the emergence of community-acquired methicillin-resistant Staphylococcus aureus. We collected data on all patients with acute osteomyelitis caused by this germ over a period of 21 years (January 1995-December 2016) and we analyzed the peculiarities of this disorder. Our case series includes 15 children, with an average age of 9 years. All patients had affected lower limb. Local trauma was reported in 8 cases and skin carriage in 4 cases. Acute onset was reported in 12 cases associated with pseudo-paralysis of the affected limb. One patient had Staphylococcus aureus pulmonary infection with signs of septicopyemia. Blood culture was positive in 8 cases. In one case PCR assay for detection of Panton-Valentine leukocidin was performed with positive result. All these patients underwent surgical debridement and received secondarily adapted empirical antibiotic therapy. Outcome was good in 8 cases and poor in the other cases, with transition to a chronic state in 6 cases and one case of death. Pathological fracture was reported in 3 cases. Osteomyelitis cause by community-acquired methicillin-resistant Staphylococcus aureus is associated with a pejorative outcome. Recognizing the clinical and paraclinical signs of these infections is essential for a specific and early therapeutic management., Competing Interests: Les auteurs ne déclarent aucun conflit d´intérêts., (Copyright: Mohamed Ben Jemaa et al.)

We sought to determine the relative value of conventional molecular methods and whole-genome sequencing (WGS) for subtyping Salmonella enterica serovar Enteritidis recovered from 2000 to 2015 in Tunisia and to investigate the genetic diversity of this serotype. A total of 175 Salmonella Enteritidis isolates were recovered from human, animal, and foodborne outbreak samples. Pulsed-field gel electrophoresis (PFGE), multiple locus variable-number tandem repeat analysis (MLVA), and whole-genome sequencing were performed. Eight pulsotypes were detected for all isolates with PFGE (DI = 0.518). Forty-five Salmonella Enteritidis isolates were selected for the MLVA and WGS techniques. Eighteen MLVA profiles were identified and classified into two major clusters (DI = 0.889). Core genome multilocus typing (cgMLST) analysis revealed 16 profiles (DI = 0.785). Whole-genome analysis indicated 660 single-nucleotide polymorphism (SNP) divergences dividing these isolates into 43 haplotypes (DI = 0.997). The phylogenetic tree supported the classification of Salmonella Enteritidis isolates into two distinct lineages subdivided into five clades and seven subclades. Pairwise SNP differences between the isolates ranged between 302 and 350. We observed about 311 SNP differences between the two foodborne outbreaks, while only less or equal to 4 SNP differences within each outbreak. SNP-based WGS typing showed an excellent discriminatory power comparing with the conventional methods such as PFGE and MLVA. Besides, we demonstrate the added value of WGS as a complementary subtyping method to discriminate outbreak from non-outbreak isolates belonging to common subtypes. It is important to continue the survey of Salmonella Enteritidis lineages in Tunisia using WGS.

Typhimurium is one of the main Salmonella serovar responsible for non-typhoidal gastro-enteritis in Tunisia. Here, we aimed to assess the genetic diversity of 88 clinical Salmonella Typhimurium strains recovered during 14 years from 2000 to 2013. Phage typing, CRISPR polymorphisms (CRISPOL), pulsed-field gel electrophoresis (PFGE), multi-locus variable-number tandem repeat analysis (MLVA) and Whole genome sequencing (WGS) were used to study the relatedness and spatio-temporal evolution of Salmonella Typhimurium populations (Typhimurium (n = 81), monophasic (n = 3) and nonmotile (n = 4) variants). Seven-locus MLST from whole genome assemblies showed that all isolates, except one, belonged to ST19. The isolates were divided into 10 definitive phage (DT) types, dominated by DT104-L (39.8%), DT41 (14.8%), DT116 (11.4%) and DT120 (5.7%). Fifty-seven MLVA patterns (DI, 0.978) were obtained compared to 11 different CRISPOL types and 15 PFGE types (DI,0.845). For cgMLST analysis, 20 profiles were found. A total of 3056 SNPs were identified from the whole genome of the 88 Salmonella Typhimurium isolates. These SNPs resolved these isolates into 86 SNP haplotypes. The phylogeny result allocated most Salmonella Typhimurium isolates into four distinct clades and seven subclades. Genetic diversity between the four clades ranged in the order of 249 to 720 nucleotide changes. The prevalent phage type DT104L formed a major clade on the phylogenetic tree. Pairwise SNP differences between the strains of this clade ranged between 0 and 59. SNP-based WGS typing seems to be the most valuable molecular markers for studying the evolutionary relationships of homogeneous serovar Typhimurium isolates., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Four group A streptococcus (GAS) bacteraemia occurred in a small burn unit within 2 weeks. The GAS patient isolates, characterized as emm89, shared the same PFGE pulsotype with two other strains isolated 2 months later. The outbreak investigation revealed that a nurse was the most likely source of GAS transmission, as she was confirmed to carry the same outbreak strain in her throat and had direct and regular contact with the six outbreak patients in the unit. The outbreak was controlled after the nurse had undergone eradication treatment. This report highlights the emergence of the emm89 clone and its capacity to elicit invasive GAS outbreaks.

Objectives: To analyze the serotype distribution of Streptococcus pneumoniae clinical isolates collected in the south of Tunisia over a 5-year period in different age groups and to assess their antimicrobial susceptibility patterns., Methods: A total of 305 non-duplicate S. pneumoniae isolates were collected between January 2012 and December 2016 at the university hospital in Sfax, Tunisia. All isolates were serotyped by multiplex PCR. The antibiotic susceptibility of all isolates was determined using the disk diffusion test or Etest assay., Results: Among the 305 pneumococcal isolates, 76 (24.9%) were invasive and 229 (75.1%) were non-invasive. The most common serotypes were 19F (20%), 14 (16.7%), 3 (9.2%), 23F (7.5%), 19A (5.9%), and 6B (5.9%). Potential immunization coverage rates for pneumococcal conjugate vaccines PCV7, PCV10, and PCV13 were 58%, 59.3%, and 78.7%, respectively. Three-quarters (75.3%) of pneumococcal isolates were non-susceptible to penicillin. The resistance rate to erythromycin was 71.4%. Only two isolates were resistant to levofloxacin., Conclusions: 19F and 14 were the most prevalent serotypes in the south of Tunisia. The inclusion of a PCV in the immunization program could be useful for reducing the burden of pneumococcal diseases. The high resistance rate to penicillin and macrolides is alarming. Prudent use of antibiotics is crucial to prevent the selection of multidrug-resistant pneumococci., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Enteritidis, Typhimurium and Livingstone are the main Salmonella enterica serovars recovered in Tunisia. Here, we aimed to assess the genetic diversity of fifty-seven Salmonella enterica strains from different sampling periods, origins and settings using pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST) and multi-locus variable-number tandem repeat analysis (MLVA). Salmonella Enteritidis, isolated from human and food sources from two regions in Sfax in 2007, were grouped into one cluster using PFGE. However, using MLVA these strains were divided into two clusters. Salmonella Typhimurium strains, recovered in 2012 and represent sporadic cases of human clinical isolates, were included in one PFGE cluster. Nevertheless, the MLVA technique, divided Salmonella Typhimurium isolates into six clusters with diversity index reaching (DI = 0.757). For Salmonella Livingstone which was responsible of two nosocomial outbreaks during 2000-2003, the PFGE and MLVA methods showed that these strains were genetically closely related. Salmonella Enteritidis and Salmonella Livingstone populations showed a single ST lineage ST11 and ST543 respectively. For Salmonella Typhimurium, two MLST sequence types ST19 and ST328 were defined. Salmonella Enteritidis and Salmonella Typhimurium strains were clearly differentiated by MLVA which was not the case using PFGE., (© 2015 APMIS. Published by John Wiley & Sons Ltd.)

Here we discuss the efficacy of colistin-tigecycline combination in the treatment of multidrug-resistant (MDR) Klebsiella pneumoniae infective endocarditis (IE). We report a case of a 67-year-old head-injured patient who developed a carbapenem-resistant K. pneumoniae IE. The patient was treated with colistin-tigecycline combination, with a favourable outcome. In conclusion, colistin-tigecycline combination may be a possible combination in the therapy of IE caused by MDR Enterobacteriaceae., (Copyright © 2015 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Objective: The aim of this study was to describe the epidemiological characteristics of Acinetobacter baumannii ventilator-associated pneumonia (VAP) and to identify factors predictive of a poor outcome., Methods: A retrospective study was conducted over 16 months in a Tunisian intensive care unit (ICU). All adult patients with A. baumannii VAP were included., Results: Ninety-two patients were included in they study; 41 (44.6%) were admitted because of multiple trauma. The mean age of the patients was 44.5±19.5 years. All patients needed mechanical ventilation on admission. The mean SAPS II score was 39±15. The mean delay before VAP onset was 8.1±4.7 days. On VAP onset, 57 patients (62%) developed septic shock. Only 14.2% of isolated strains were susceptible to imipenem; none were resistant to colistin. The mean duration of mechanical ventilation was 20±11 days. The mean duration of ICU stay was 24.3±18.7 days. ICU mortality was 60.9%. In the multivariate analysis, factors predictive of a poor outcome were previously known hypertension (odds ratio 5.8, 95% confidence interval 1.4-24.9; p=0.018) and VAP-related septic shock (odds ratio 8.5, 95% confidence interval 3-23.7; p<0.001)., Conclusion: A. baumannii VAP is associated with a high mortality. Hemodynamic impairment is predictive of a poor outcome., (Copyright © 2013 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: Extended-spectrum β-lactamases (ESBLs), particularly CTX-M- type ESBLs, are among the most important resistance determinants spreading worldwide in Enterobacteriaceae. The aim of this study was to characterize a collection of 163 ESBL-producing Escherichia coli collected in Tunisia, their ESBL-encoding plasmids and plasmid associated addiction systems., Results: The collection comprised 163 ESBL producers collected from two university hospitals of Sfax between 1989 and 2009. 118 isolates harbored blaCTX-M gene (101 blaCTX-M-15 gene and 17 blaCTX-M-14 gene). 49 isolates carried blaSHV-12 gene, 9 blaSHV-2a gene and only 3 blaTEM-26 gene. 16 isolates produced both CTX-M and SHV-12. The 101 CTX-M-15-producing isolates were significantly associated to phylogroup B2 and exhibiting a high number of virulence factors. 24 (23.7%) of the group B2 isolates belonged to clonal complex ST131. Pulsed-field gel electrophoresis (PFGE) typing revealed a genetic diversity of the isolates. 144 ESBL determinants were transferable mostly by conjugation. The majority of plasmid carrying blaCTX-M-15 genes (72/88) were assigned to various single replicon or multireplicon IncF types and had significantly a higher frequency of addiction systems, notably the VagCD module., Conclusion: This study demonstrates that the dissemination of CTX-M-15 producing E. coli in our setting was due to the spread of various IncF-type plasmids harboring multiple addiction systems, into related clones with high frequency of virulence determinants.

Objectives: To report an outbreak due to Providencia stuartii isolates carrying bla(OXA-48), bla(PER-1), bla(CMY-4) and qnrA6 in a Tunisian hospital in 2011., Methods: Eight intensive care unit (ICU) patients infected/colonized by extended-spectrum β-lactamase (ESBL)-producing P. stuartii between March and May 2011 were included. Molecular epidemiology was studied by PFGE. Antibiotic resistance genes were analysed by PCR and sequencing and the plasmid incompatibility group by a PCR-based replicon typing scheme., Results: Eight patients were colonized with ESBL-producing P. stuartii isolates. All these isolates were clonally related and found to carry bla(OXA-48), bla(PER-1), bla(CMY-4), qnrA6 and aac-6'-Ib genes on the same self-conjugative IncA/C plasmid. The same strain was also cultured from environmental samples in the ICU. All these isolates were susceptible to carbapenems. Only one colonized patient developed P. stuartii pleurisy and was effectively treated with imipenem alone., Conclusions: This is the first report of an outbreak due to P. stuartii isolates carrying bla(OXA-48) in Tunisia. The simultaneous expression of various resistance genes (bla(OXA-48), bla(CMY-4), bla(PER-1), qnrA and aac-6'-Ib) by P. stuartii isolates is alarming.

Twenty-three strains of Staphylococcus aureus with borderline resistance to oxacillin were studied. These strains were not detected by the cefoxitin test, tests for penicillin-binding protein 2a (PBP2a), mecA, and mecA(LGA251) were negative, and the strains were genetically unrelated. To detect all strains resistant to oxacillin, laboratories should routinely test for both cefoxitin and oxacillin.

This study was conducted to identify the β-lactamase content of 30 metallo-β-lactamase-producing Pseudomonas aeruginosa isolated in 2007 from two Tunisian hospitals and to investigate their genetic relatedness. All these isolates produced VIM-2. bla(PER-1), bla(PSE-1), bla(OXA-2), and bla(OXA-10) were identified in 17, 5, 21, and 1 isolates, respectively. These enzymes were often associated in the same isolate: 26 isolates had at least two β-lactamases. The predominant serotype was O12. Pulsed-field gel electrophoresis revealed genetic diversity among the metallo-β-lactamase-producing P. aeruginosa isolates. This is the first report on the existence of bla(PER-1), bla(PSE-1), bla(OXA-2), and bla(OXA-10) in Tunisia., (© Mary Ann Liebert, Inc.)

Objectives: Escherichia coli producing CTX-M-type extended-spectrum beta-lactamases (ESBLs) are spreading worldwide. The aim of this work was to investigate the addiction systems carried by the replicons involved in the emergence and spread of ESBLs in relation to ESBL and replicon types., Methods: A collection of 125 TEM, SHV and CTX-M ESBL-producing E. coli isolates and their 125 transconjugants or transformants was analysed. Five plasmid protein antitoxin-regulated systems and three plasmid antisense RNA-regulated systems were sought by PCR., Results: Two hundred and ninety-eight plasmid addiction systems were detected in the parental strains (mean 2.38, range 0-6 per strain) and 86 were detected in the recipient strains (mean 0.69, range 0-5 per strain). PemKI, CcdAB, Hok-Sok and VagCD were the most frequently represented systems in both recipient and parental strains. The parental SHV and CTX-M ESBL-producing strains had more addiction systems than the TEM ESBL producers. In the recipient strains, the frequency of addiction systems was significantly higher in IncF plasmids. Among the IncF replicons carrying CTX-M-type enzymes, the frequency of addiction systems was significantly higher in IncF plasmids carrying CTX-M-15 (mean 3.5) or CTX-M-9 (mean 4) than in those carrying CTX-M-14 (mean 0.6)., Conclusions: In E. coli producing CTX-M-15 or CTX-M-9 ESBLs, plasmids bearing the bla(CTX-M) gene have multiple addiction systems that could contribute to their maintenance in host strains.

Unlabelled: THE AIM of the study was to type Serratia marcescens responsible for nosocomial outbreaks in an intensive care unit in Sfax -Tunisia., Methods: The relatedness between S. marcescens isolates was studied by Pulsed field gel electrophoresis (PFGE). We included 56 strains of Serratia marcescens isolated from patients hospitalized in the intensive care unit during 2003 and 2004. Seven epidemiological unrelated strains of Serratia marcescens were also tested. Samples from environment and hands of the nursing and medical staff were collected and cultured to identify the source of contamination., Results: All strains showed a wild type of antimicrobial susceptibility. PFGE typing revealed that three different clones were present. None of the cultures taken from hands of unit staff and from environmental samples yielded positive results for S. marcescens., Conclusion: We have confirmed the presence of three consecutive outbreaks caused by three genetically unrelated bacterial clones of Serratia marcescens in the intensive care unit ward. These outbreaks are closely related to the frequent use of colistin and the lack of measures of hygiene in this ward.

Klebsiella pneumoniae clinical isolates resistant to carbapenems were recovered from 11 patients in the hospital of Sfax, Tunisia. The isolates were closely related as shown by pulsed-field gel electrophoresis, and they produced VIM-4 metallo-enzyme, CTX-M-15 extended-spectrum beta-lactamase, and CMY-4 AmpC enzyme. The bla(VIM-4) gene is part of a class 1 integron.

We report the third case of endocarditis caused by the newly described micrococcal species Kytococcus schroeteri. A 49-year-old woman was admitted to the hospital with suspected prosthetic valve endocarditis. Five blood cultures and prosthetic valve cultures grew the same type of organism, initially identified as Micrococcus sp. Assignment to the genus Kytococcus was suggested by the arginine dihydrolase activity and resistance to oxacillin. After sequencing of the 16S rRNA genes, the isolate was recognized as K. schroeteri. The patient was treated first with vancomycin combined with gentamicin and later with pristinamycin and rifampin. Three cases of K. schroeteri endocarditis described within a short period of time might indicate a specific pathogenicity of this new species. The isolation of kytococci from normally sterile sites should not be overlooked.