Shotgun Proteomic-Based Approach with a Q-Exactive Hybrid Quadrupole-Orbitrap High-Resolution Mass Spectrometer for Protein Adductomics on a 3D Human Brain Tumor Neurospheroid Culture Model: The Identification of Adduct Formation in Calmodulin-Dependent Protein Kinase-2 and Annexin-A1 Induced by Pesticide Mixture.

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.

Research of Pesticide Metabolites in Human Brain Tumor Tissues by Chemometrics-Based Gas Chromatography-Mass Spectrometry Analysis for a Hypothetical Correlation between Pesticide Exposure and Risk Factor of Central Nervous System Tumors.

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.

Differential Proteome Profiling Analysis under Pesticide Stress by the Use of a Nano-UHPLC-MS/MS Untargeted Proteomic-Based Approach on a 3D-Developed Neurospheroid Model: Identification of Protein Interactions, Prognostic Biomarkers, and Potential Therapeutic Targets in Human IDH Mutant High-Grade Gliomas.

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.

Antibiotic resistance pattern, capsular types, and molecular characterization of invasive isolates of Streptococcus pneumoniae in the south of Tunisia from 2012 to 2018.

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 ß-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.

Antimicrobial resistance genes, virulence markers and prophage sequences in Salmonella enterica serovar Enteritidis isolated in Tunisia using whole genome sequencing.

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.

Prevalence of Helicobacter pylori resistance to clarithromycin in Tunisia.

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.

Trends in human intestinal carriage of ESBL- and carbapenemase-producing Enterobacterales among food handlers in Tunisia: emergence of C1-M27-ST131 subclades, blaOXA-48 and blaNDM.

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 ß-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.

Relationship Between Serotypes and Antimicrobial Nonsusceptibility of Streptococcus pneumoniae Clinical Isolates in Tunisia.

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.

[Acute osteomyelitis caused by community-acquired methicillin-resistant Staphylococcus aureus in children: about 15 cases]. / L´ostéomyélite aiguë à Staphylocoque aureus résistant à la méthicilline d´origine communautaire chez l´enfant: à propos de 15 cas.

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.

Clinical optochin resistant Streptococcus pneumoniae and Streptococcus pseudopneumoniae strains in Tunisia.

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.

Comparison of conventional molecular and whole-genome sequencing methods for subtyping Salmonella enterica serovar Enteritidis strains from Tunisia.

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.

Genetic diversity of clinical Salmonella enterica serovar Typhimurium in a university hospital of south Tunisia, 2000-2013.

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.

A report on the first outbreak of emm89 group A streptococcus invasive infections in a burns unit in Tunisia.

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.

Serotype distribution and antibiotic susceptibility of Streptococcus pneumoniae strains in the south of Tunisia: A five-year study (2012-2016) of pediatric and adult populations.

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.

Molecular epidemiological characteristics of Salmonella enterica serovars Enteritidis, Typhimurium and Livingstone strains isolated in a Tunisian university hospital.

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.