The α3 subunit of GABAA receptors promotes formation of inhibitory synapses in the absence of collybistin.

Signaling at nerve cell synapses is a key determinant of proper brain function, and synaptic defects-or synaptopathies-are at the basis of many neurological and psychiatric disorders. Collybistin (CB), a brain-specific guanine nucleotide exchange factor, is essential for the formation of γ-aminobutyric acidergic (GABAergic) postsynapses in defined regions of the mammalian forebrain, including the hippocampus and basolateral amygdala. This process depends on a direct interaction of CB with the scaffolding protein gephyrin, which leads to the redistribution of gephyrin into submembranous clusters at nascent inhibitory synapses. Strikingly, synaptic clustering of gephyrin and GABAA type A receptors (GABAARs) in several brain regions, including the cerebral cortex and certain thalamic areas, is unperturbed in CB-deficient mice, indicating that the formation of a substantial subset of inhibitory postsynapses must be controlled by gephyrin-interacting proteins other than CB. Previous studies indicated that the α3 subunit of GABAARs (GABAAR-α3) binds directly and with high affinity to gephyrin. Here, we provide evidence (i) that a homooligomeric GABAAR-α3A343W mutant induces the formation of submembranous gephyrin clusters independently of CB in COS-7 cells, (ii) that gephyrin clustering is unaltered in the neuronal subpopulations endogenously expressing the GABAAR-α3 in CB-deficient brains, and (iii) that exogenous expression of GABAAR-α3 partially rescues impaired gephyrin clustering in CB-deficient hippocampal neurons. Our results identify an important role of GABAAR-α3 in promoting gephyrin-mediated and CB-independent formation of inhibitory postsynapses.

A GTPase-induced switch in phospholipid affinity of collybistin contributes to synaptic gephyrin clustering.

Synaptic transmission between neurons relies on the exact spatial organization of postsynaptic transmitter receptors, which are recruited and positioned by dedicated scaffolding and regulatory proteins. At GABAergic synapses, the regulatory protein collybistin (Cb, also known as ARHGEF9) interacts with small GTPases, cell adhesion proteins and phosphoinositides to recruit the scaffolding protein gephyrin and GABAA receptors to nascent synapses. We dissected the interaction of Cb with the small Rho-like GTPase TC10 (also known as RhoQ) and phospholipids. Our data define a protein-lipid interaction network that controls the clustering of gephyrin at synapses. Within this network, TC10 and monophosphorylated phosphoinositides, particulary phosphatidylinositol 3-phosphate (PI3P), provide a coincidence detection platform that allows the accumulation and activation of Cb in endomembranes. Upon activation, TC10 induces a phospholipid affinity switch in Cb, which allows Cb to specifically interact with phosphoinositide species present at the plasma membrane. We propose that this GTPase-based regulatory switch mechanism represents an important step in the process of tethering of Cb-dependent scaffolds and receptors at nascent postsynapses.

Neuroligin-2 dependent conformational activation of collybistin reconstituted in supported hybrid membranes.

The assembly of the postsynaptic transmitter sensing machinery at inhibitory nerve cell synapses requires the intimate interplay between cell adhesion proteins, scaffold and adaptor proteins, and γ-aminobutyric acid (GABA) or glycine receptors. We developed an in vitro membrane system to reconstitute this process, to identify the essential protein components, and to define their mechanism of action, with a specific focus on the mechanism by which the cytosolic C terminus of the synaptic cell adhesion protein Neuroligin-2 alters the conformation of the adaptor protein Collybistin-2 and thereby controls Collybistin-2-interactions with phosphoinositides (PtdInsPs) in the plasma membrane. Supported hybrid membranes doped with different PtdInsPs and 1,2-dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} nickel salt (DGS-NTA(Ni)) to allow for the specific adsorption of the His6-tagged intracellular domain of Neuroligin-2 (His-cytNL2) were prepared on hydrophobically functionalized silicon dioxide substrates via vesicle spreading. Two different collybistin variants, the WT protein (CB2SH3) and a mutant that adopts an intrinsically 'open' and activated conformation (CB2SH3/W24A-E262A), were bound to supported membranes in the absence or presence of His-cytNL2. The corresponding binding data, obtained by reflectometric interference spectroscopy, show that the interaction of the C terminus of Neuroligin-2 with Collybistin-2 induces a conformational change in Collybistin-2 that promotes its interaction with distinct membrane PtdInsPs.

Collybistin SH3-protein isoforms are expressed in the rat brain promoting gephyrin and GABA-A receptor clustering at GABAergic synapses.

Collybistin (CB) is a guanine nucleotide exchange factor (GEF) selectively localized at GABAergic and glycinergic postsynapses. Analysis of mRNA shows that several isoforms of collybistin are expressed in the brain. Some of the isoforms have a SH3 domain (CBSH3+) and some have no SH3 domain (CBSH3-). The CBSH3+ mRNAs are predominantly expressed over CBSH3-. However, in an immunoblot study of mouse brain homogenates, only CBSH3+ protein isoforms were detected, proposing that CBSH3- protein might not be expressed in the brain. The expression or lack of expression of CBSH3- protein is an important issue because CBSH3- has a strong effect in promoting the postsynaptic clustering of gephyrin and GABA-A receptors (GABAA Rs). Moreover CBSH3- is constitutively active; therefore lower expression of CBSH3- protein might play a relatively stronger functional role than the more abundant but self-inhibited CBSH3+ isoforms, which need to be activated. We are now showing that: (a) CBSH3- protein is expressed in the brain; (b) parvalbumin positive (PV+) interneurons show higher expression of CBSH3- protein than other neurons; (c) CBSH3- is associated with GABAergic synapses in various regions of the brain and (d) knocking down CBSH3- in hippocampal neurons decreases the synaptic clustering of gephyrin and GABAA Rs. The results show that CBSH3- protein is expressed in the brain and that it plays a significant role in the size regulation of the GABAergic postsynapse.

The health and economic impact of acute gastroenteritis in Belgium, 2010-2014.

Acute gastroenteritis (AGE) remains a common condition in both low- and high-income countries. In Belgium, however, there is currently a lack of information on the societal health and economic impact of AGE. We conducted a retrospective study using mortality and cause-of-death data, hospital data, primary care data, health interview survey data and other published data. We estimated the burden of illness during a 5-year period (2010-2014) in Belgium in terms of deaths, patients admitted to hospitals, patients consulting their general practitioner (GP) and cases occurring in the community. We further quantified the health impact in terms of disability-adjusted life years (DALYs) and the economic impact in terms of cost-of-illness estimates. We estimated 343 deaths, 27 707 hospitalised patients, 464 222 GP consultations and 10 058 741 episodes occurring in the community (0.91 cases/person) on average per year. AGE was associated with 11 855 DALYs per year (107 DALY per 100 000 persons). The economic burden was estimated to represent direct costs of €112 million, indirect costs of €927 million (90% of the total costs) and an average total cost of €103 per case and €94 per person. AGE results in a substantial health and economic impact in Belgium, justifying continued mitigation efforts.

Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) in bulk tank milk, livestock and dairy-farm personnel in north-central and north-eastern Greece: Prevalence, characterization and genetic relatedness.

Recently, there has been an increased tendency towards raw-milk consumption, which may pose a consumer risk, due to the possible presence of human pathogenic microorganisms, such as Staphylococcus aureus and even methicillin-resistant S. aureus (MRSA). The prevalence of S. aureus and methicillin-resistant S. aureus (MRSA) was investigated in 40 dairy (cattle, sheep and goat) farms in northern Greece. S. aureus and MRSA were detected in 47.8% and 4.1% of the 387 samples (raw milk, farmers and animal samples) tested, respectively. Most (81.3%) of the MRSA isolates harbored the mecA gene, whereas the mecC or Panton-Valentine Leucocidin (PVL) genes were not detected. Seven spa types were identified, with t127 being the most prevalent. Spa type t034 (CC398) was isolated for the first time from livestock in Greece. Staphylococcal enterotoxin genes were detected in 93.8% of the MRSA isolates. The MRSA isolates were genetically diverse and were all capable of biofilm production. Our results confirm the lurking threat of MRSA in raw milk and dairy farms and suggest the need for surveillance programs starting at the farm level.

Endosomal Phosphatidylinositol 3-Phosphate Promotes Gephyrin Clustering and GABAergic Neurotransmission at Inhibitory Postsynapses.

The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the proper assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic postsynapses requires the scaffold protein gephyrin and the guanine nucleotide exchange factor collybistin (Cb). In vitro, the pleckstrin homology domain of Cb binds phosphoinositides, specifically phosphatidylinositol 3-phosphate (PI3P). However, whether PI3P is required for inhibitory postsynapse formation is currently unknown. Here, we investigated the role of PI3P at developing GABAergic postsynapses by using a membrane-permeant PI3P derivative, time-lapse confocal imaging, electrophysiology, as well as knockdown and overexpression of PI3P-metabolizing enzymes. Our results provide the first in cellula evidence that PI3P located at early/sorting endosomes regulates the postsynaptic clustering of gephyrin and GABAA receptors and the strength of inhibitory, but not excitatory, postsynapses in cultured hippocampal neurons. In human embryonic kidney 293 cells, stimulation of gephyrin cluster formation by PI3P depends on Cb. We therefore conclude that the endosomal pool of PI3P, generated by the class III phosphatidylinositol 3-kinase, is important for the Cb-mediated recruitment of gephyrin and GABAA receptors to developing inhibitory postsynapses and thus the formation of postsynaptic membrane specializations.

A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.

The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic and glycinergic synapses is controlled by the scaffold protein gephyrin and the adaptor protein collybistin. We derived new insights into the structure of collybistin and used these to design biochemical, cell biological, and genetic analyses of collybistin function. Our data define a collybistin-based protein interaction network that controls the gephyrin content of inhibitory postsynapses. Within this network, collybistin can adopt open/active and closed/inactive conformations to act as a switchable adaptor that links gephyrin to plasma membrane phosphoinositides. This function of collybistin is regulated by binding of the adhesion protein neuroligin-2, which stabilizes the open/active conformation of collybistin at the postsynaptic plasma membrane by competing with an intramolecular interaction in collybistin that favors the closed/inactive conformation. By linking trans-synaptic neuroligin-dependent adhesion and phosphoinositide signaling with gephyrin recruitment, the collybistin-based regulatory switch mechanism represents an integrating regulatory node in the formation and function of inhibitory postsynapses.

Prevalence of Staphylococcus aureus and of methicillin-resistant S. aureus (MRSA) along the production chain of dairy products in north-western Greece.

The objective of this study was to estimate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in the production chain of dairy products. Of 367 tested samples (36 bulk tank milk (BTM), 19 dairy products, 72 human, 185 animal, 55 equipment), 212 (57.8%) were found positive for S. aureus. Almost all isolates (99.6%) were resistant to at least one antimicrobial and 13.3% were multi-drug resistant (MDR), exhibiting resistance to three or more antibiotic classes. Eleven samples (3%) were found contaminated by MRSA carrying the mecA gene. None of the MRSA isolates carried the mecC or the Pandon-Valentine leucocidin (PVL) genes. Four spa types were identified among the MRSA isolates: t127, t3586, t1773, t4038, with t127 being the most prevalent (7 out of 11). Two of them, t3586 and t1773, were isolated for the first time in Greece. Furthermore, Pulse-Field Gel Electrophoresis (PFGE) analysis indicated clonal circulation through the dairy production chain. The presence of MDR S. aureus, and especially MRSA, in animals and dairy products represents a potential threat for the spread of this pathogen in the community. The results indicated that human, animal and environmental sources could be involved in the contamination of dairy products along their production chain and therefore further investigation of contamination sources is needed to control the dispersion of MRSA in the community.

Specificity of Collybistin-Phosphoinositide Interactions: IMPACT OF THE INDIVIDUAL PROTEIN DOMAINS.

The regulatory protein collybistin (CB) recruits the receptor-scaffolding protein gephyrin to mammalian inhibitory glycinergic and GABAergic postsynaptic membranes in nerve cells. CB is tethered to the membrane via phosphoinositides. We developed an in vitro assay based on solid-supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes doped with different phosphoinositides on silicon/silicon dioxide substrates to quantify the binding of various CB2 constructs using reflectometric interference spectroscopy. Based on adsorption isotherms, we obtained dissociation constants and binding capacities of the membranes. Our results show that full-length CB2 harboring the N-terminal Src homology 3 (SH3) domain (CB2SH3+) adopts a closed and autoinhibited conformation that largely prevents membrane binding. This autoinhibition is relieved upon introduction of the W24A/E262A mutation, which conformationally "opens" CB2SH3+ and allows the pleckstrin homology domain to properly bind lipids depending on the phosphoinositide species with a preference for phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate. This type of membrane tethering under the control of the release of the SH3 domain of CB is essential for regulating gephyrin clustering.

IQ Motif and SEC7 Domain-containing Protein 3 (IQSEC3) Interacts with Gephyrin to Promote Inhibitory Synapse Formation.

Gephyrin is a central scaffold protein that mediates development, function, and plasticity of mammalian inhibitory synapses by interacting with various inhibitory synaptic proteins. Here, we show that IQSEC3, a guanine nucleotide exchange factor for ARF6, directly interacts with gephyrin, an interaction that is critical for the inhibitory synapse localization of IQSEC3. Overexpression of IQSEC3 increases inhibitory, but not excitatory, synapse density in a guanine nucleotide exchange factor activity-dependent manner. Conversely, knockdown of IQSEC3 decreases size of gephyrin cluster without altering gephyrin puncta density. Collectively, these data reveal that IQSEC3 acts together with gephyrin to regulate inhibitory synapse development.

Differences in sheep and goats milk microbiological profile between conventional and organic farming systems in Greece.

The aim of this study was to examine differences in the microbiological profile and antimicrobial resistance of bacteria isolated from milk from organic and conventional sheep and goat farms. Twenty-five organic and 25 conventional sheep and goat farms in the region of Thessaly, Greece participated in this study. A standardised detailed questionnaire was used to describe farming practices. A total of 50 samples were collected and analysed for total viable count (TVC), total coliform count (TCC) and somatic cell count (SCC), while Staphylococcus aureus and Escherichia coli were isolated using standard methods. Isolates were identified at species level by Api-test and Matrix-Assisted Laser Desorption/Ionisation-Time of Flight Mass Spectrometry (MALDI-TOF MS). Susceptibility to a panel of 20 for E. coli and 16 for S. aureus antimicrobials was determined by the agar dilution method. Pulsed Field Gel Electrophoresis (PFGE) was performed for S. aureus and E. coli isolates to determine predominant clones. Lower counts of TVC, TCC and SCC were identified in milk from the organic farms, possibly due to differences in the hygienic farming practices found on those farms. API-tests and MALDI-TOF MS showed no significant differences in the S. aureus and E. coli isolates. Overall, antimicrobial resistance rates were low, while a statistically higher percentage was estimated among strains originating from conventional farms in comparison with organic farms, possibly due to the restriction of antibiotic use in organic farming. PFGE revealed diversity among S. aureus and E. coli populations in both organic and conventional farms indicating circulation of 2-3 main clones changing slightly during their evolution. Consequently, there is evidence that milk from the organic farms presents a better microbiological profile when compared with milk from conventional farms.

Lipid binding defects and perturbed synaptogenic activity of a Collybistin R290H mutant that causes epilepsy and intellectual disability.

Signaling at nerve cell synapses is a key determinant of proper brain function, and synaptic defects--or synaptopathies--are at the basis of many neurological and psychiatric disorders. In key areas of the mammalian brain, such as the hippocampus or the basolateral amygdala, the clustering of the scaffolding protein Gephyrin and of γ-aminobutyric acid type A receptors at inhibitory neuronal synapses is critically dependent upon the brain-specific guanine nucleotide exchange factor Collybistin (Cb). Accordingly, it was discovered recently that an R290H missense mutation in the diffuse B-cell lymphoma homology domain of Cb, which carries the guanine nucleotide exchange factor activity, leads to epilepsy and intellectual disability in human patients. In the present study, we determined the mechanism by which the Cb(R290H) mutation perturbs inhibitory synapse formation and causes brain dysfunction. Based on a combination of biochemical, cell biological, and molecular dynamics simulation approaches, we demonstrate that the R290H mutation alters the strength of intramolecular interactions between the diffuse B-cell lymphoma homology domain and the pleckstrin homology domain of Cb. This defect reduces the phosphatidylinositol 3-phosphate binding affinity of Cb, which limits its normal synaptogenic activity. Our data indicate that impairment of the membrane lipid binding activity of Cb and a consequent defect in inhibitory synapse maturation represent a likely molecular pathomechanism of epilepsy and mental retardation in humans.

Collybistin activation by GTP-TC10 enhances postsynaptic gephyrin clustering and hippocampal GABAergic neurotransmission.

In many brain regions, gephyrin and GABAA receptor clustering at developing inhibitory synapses depends on the guanine nucleotide exchange factor collybistin (Cb). The vast majority of Cb splice variants contain an autoinhibitory src homology 3 domain, and several synaptic proteins are known to bind to this SH3 domain and to thereby activate gephyrin clustering. However, many functional GABAergic synapses form independently of the known Cb-activating proteins, indicating that additional Cb activators must exist. Here we show that the small Rho-like GTPase TC10 stimulates Cb-dependent gephyrin clustering by binding in its active, GTP-bound state to the pleckstrin homology domain of Cb. Overexpression of a constitutively active TC10 variant in neurons causes an increase in the density of synaptic gephyrin clusters and mean miniature inhibitory postsynaptic current amplitudes, whereas a dominant negative TC10 variant has opposite effects. The enhancement of Cb-induced gephyrin clustering by GTP-TC10 does not depend on the guanine nucleotide exchange activity of Cb but involves an interaction that resembles reported interactions of other small GTPases with their effectors. Our data indicate that GTP-TC10 activates the major src homology 3 domain-containing Cb variants by relieving autoinhibition and thus define an alternative GTPase-driven signaling pathway in the genesis of inhibitory synapses.

Comparison of CE-MS/MS and LC-MS/MS sequencing demonstrates significant complementarity in natural peptide identification in human urine.

Clinical proteomics has led to the identification of biomarkers specifically associated with a clinical condition that can serve for diagnostic or prognostic purposes. Learning more about the origin of these protein fragments would lead to a better insight in the pathology, and this requires improved identification of the peptide sequences. The aim of this study is to assess the complementarity of LC-MS/MS and CE-MS/MS as techniques in peptide sequence identification of the urinary low-molecular weight proteome. A male standard human urine sample was analyzed using LC- and CE-MS/MS (n = 10 per technique), identifying 905 unique peptide sequences with high confidence, 50% of those were identified only with LC, 20% only with CE and 30% with both techniques. Higher LC coverage might be due in part to the higher amount of sample that can be loaded onto an LC column. Peptides uniquely identified in CE are generally small and highly charged, likely unable to bind to the LC column In conclusion, we showed that LC-MS/MS and CE-MS/MS are highly complementary in identifying peptide sequences. The combination of both technologies results in significantly increased sequence coverage.

Homeostatic regulation of gephyrin scaffolds and synaptic strength at mature hippocampal GABAergic postsynapses.

Gephyrin is a scaffolding protein important for the postsynaptic clustering of inhibitory neurotransmitter receptors. Here, we investigated the properties of gephyrin scaffolds at γ-aminobutyric acid- (GABA-)ergic synapses in organotypic entorhino-hippocampal cultures prepared from a transgenic mouse line, which expresses green fluorescent protein-tagged gephyrin under the control of the Thy1.2 promoter. Fluorescence recovery after photobleaching revealed a developmental stabilization of postsynaptic gephyrin clusters concomitant with an increase in cluster size and synaptic strength between 1 and 4 weeks in vitro. Prolonged treatment of the slice cultures with diazepam or a GABAA receptor antagonist disclosed a homeostatic regulation of both inhibitory synaptic strength and gephyrin cluster size and stability in 4-weeks-old cultures, whereas at 1 week in vitro, the same drug treatments modulated GABAergic postsynapse and gephyrin cluster properties following a Hebbian mode of synaptic plasticity. Our data are consistent with a model in which the postnatal maturation of the hippocampal network endows CA1 pyramidal neurons with the ability to homeostatically adjust the strength of their inhibitory postsynapses to afferent GABAergic drive by regulating gephyrin scaffold properties.

IMAC fractionation in combination with LC-MS reveals H2B and NIF-1 peptides as potential bladder cancer biomarkers.

Improvement in bladder cancer (BC) management requires more effective diagnosis and prognosis of disease recurrence and progression. Urinary biomarkers attract special interest because of the noninvasive means of urine collection. Proteomic analysis of urine entails the adoption of a fractionation methodology to reduce sample complexity. In this study, we applied immobilized metal affinity chromatography in combination with high-resolution LC-MS/MS for the discovery of native urinary peptides potentially associated with BC aggressiveness. This approach was employed toward urine samples from patients with invasive BC, noninvasive BC, and benign urogenital diseases. A total of 1845 peptides were identified, corresponding to a total of 638 precursor proteins. Specific enrichment for proteins involved in nucleosome assembly and for zinc-finger transcription factors was observed. The differential expression of two candidate biomarkers, histone H2B and NIF-1 (zinc finger 335) in BC, was verified in independent sets of urine samples by ELISA and by immunohistochemical analysis of BC tissue. The results collectively support changes in the expression of both of these proteins with tumor progression, suggesting their potential role as markers for discriminating BC stages. In addition, the data indicate a possible involvement of NIF-1 in BC progression, likely as a suppressor and through interactions with Sox9 and HoxA1.

Increased dentate gyrus excitability in neuroligin-2-deficient mice in vivo.

The postsynaptic adhesion protein neuroligin-2 (NL2) is selectively localized at inhibitory synapses. Here, we studied network activity in the dentate gyrus of NL2-deficient mice following perforant path (PP) stimulation in vivo. We found a strong increase in granule cell (GC) excitability. Furthermore, paired-pulse inhibition (PPI) of the population spike, a measure for γ-aminobutyric acid (GABA)ergic network inhibition, was severely impaired and associated with reduced GABA(A) receptor (GABA(A)R)-mediated miniature inhibitory postsynaptic currents recorded from NL2-deficient GCs. In agreement with these functional data, the number of gephyrin and GABA(A)R clusters was significantly reduced in the absence of NL2, indicating a loss of synaptic GABA(A)Rs from the somata of GCs. Computer simulations of the dentate network showed that impairment of perisomatic inhibition is able to explain the electrophysiological changes observed in the dentate circuitry of NL2 knockout animals. Collectively, our data demonstrate for the first time that deletion of NL2 increases excitability of cortical neurons in the hippocampus of intact animals, most likely through impaired GABA(A)R clustering.

Training in tools to develop quantitative risk assessment of fresh produce using water reuse systems in Mediterranean production.

Water resources are increasingly coming under pressure specially around the Mediterranean area, leading to water scarcity and a deterioration in water quality. The use of treated wastewater represents an alternative source to enhance the demand for irrigation water. Water reuse in combination with the promotion of the use of water-efficient technologies in industry and water-saving irrigation techniques could lead to good qualitative and quantitative water status for surface and ground water bodies. Nevertheless, food-borne outbreaks linked to fresh produce irrigated with partially or untreated wastewater caused by bacteria, parasites and enteropathogenic viruses have been widely reported. In the absence of solid scientific understanding of the actual risks involved, consumers are likely less receptive to buy leafy greens irrigated with treated wastewater, also known as reclaimed water. In this study, we aimed to assess the microbiological risks of leafy green vegetables irrigated with treated wastewater in Spain using Norovirus as a model organism to facilitate the development of risk management strategies. A conceptual exposure model was designed to describe the virus fate and transport from the Wastewater treatment plant (WWTP) secondary effluent to the consumers' fork. This study is an example of the use of reclaimed water for irrigation of commercial fields producing leafy greens in the south-east of Spain and tries to assess potential microbiological risks to the consumers by establishing their safety.

Phenotypic and molecular characterization of blaCTX-M-3 and blaCTX-M-55-producing monophasic Salmonella enterica serovar Typhimurium in Greece.

OBJECTIVES: To characterize three Salmonella enterica serovar Typhimurium strains using whole genome sequencing (WGS) and conventional methods. The isolates were recovered from three pediatric patients in Greece as part of the hospital's epidemiological surveillance system during 2016 to 2018. METHODS: Bacterial identification and antimicrobial susceptibility testing was performed using the VITEK 2 automated system, disc diffusion test, and MIC gradient test while serotyping by the slide agglutination method. Detection of resistance genes, eBurst groups (eBG), assignment to sequence types, single nucleotide polymorphism (SNP) typing, location and characterization of drug resistance regions, and in silico plasmid detection were carried out using WGS. RESULTS: All strains were identified as S. Typhimurium-monophasic, ST34, eBG1 with antigenic formula 1,4, [5], 12:i:-. They were phenotypically resistant to most antibiotics tested except piperacillin/tazobactam, imipenem, and co-trimoxazole. WGS revealed the chromosomally located genes encoding the ASSuT (ampicillin, streptomycin, sulfonamides, and tetracycline) resistant pattern in all three strains. WGS revealed extended spectrum ß-lactamase (ESBL) production in all three strains, the presence of blaCTX-M-3 on an IncI1 plasmid in two strains isolated in 2018, and the chromosomally encoded blaCTX-M-55 plus qnrS1 (resistance to ciprofloxacin) in the strain isolated in 2016. The two strains from 2018 were isolated from the same hospital ward and were genetically related. CONCLUSIONS: The emergence of ESBL among S. 1,4,[5], 12:i:- is worrisome due to its increasing antimicrobial resistance phenotype, making clinical treatment difficult. WGS provides an alternative to traditional methods of identification and genomic characterisation of strains, and serves to better understand their epidemiological dynamics and bacterial pathogenesis.