River waters in Greece: A reservoir for clinically relevant extended-spectrum-ß-lactamases-producing Escherichia coli.

In the current study, the genotypic characteristics such as antimicrobial resistance and virulence genes, and plasmid replicons and phenotypic characteristics such as biofilm formation and antimicrobial resistance of 87 extended-spectrum beta-lactamase (ESBL)-producing E. coli (ESBL-Ec) isolated from 7 water bodies in northern Greece were investigated. Our data show a high prevalence (60.0 %) of ESBL-Ec in surface waters that exhibit high genetic diversity, suggesting multiple sources of their transmission into the aquatic environment. When evaluating the antimicrobial resistance of isolates, wide variation in their resistance profiles has been detected, with all isolates being multi-drug resistant (MDR). Regarding biofilm formation capacity and phylogenetic groups, the majority (54.0 %, 47/87) of ESBL-Ec were classified as no biofilm producers mainly assigned to phylogroup A (35.6 %; 31/87), followed by B2 (26.5 %; 23/87). PCR screening showed that a high proportion of the isolates tested positive for the blaCTX-M-1 group genes (69 %, 60/87), followed by blaTEM (55.2 %, 48/87), blaOXA (25.3 %, 22/87) and blaCTX-M-9 (17.2 %, 15/87). A subset of 28 ESBL-Ec strains was further investigated by applying whole genome sequencing (WGS), and among them, certain clinically significant sequence types were identified, such as ST131 and ST10. The corresponding in silico analysis predicted all these isolates as human pathogens, while a significant proportion of WGS-ESBL-Ec were assigned to extraintestinal pathogenic E. coli (ExPEC; 32.1 %), and urinary pathogenic E. coli (UPEC; 28.6 %) pathotypes. Comparative phylogenetic analysis, showed that the genomes of the ST131-O25:H4-H30 isolates are genetically linked to the human clinical strains. Here, we report for the first time the detection of a plasmid-mediated mobile colistin resistance gene in ESBL-Ec in Greece isolated from an environmental source. Overall, this study underlines the role of surface waters as a reservoir for antibiotic resistance genes and for presumptive pathogenic ESBL-Ec.

The Experience of a Tertiary Reference Hospital in the Study of Rare Neurological Diseases.

Background and Objectives: Rare diseases (RDs) are life-threatening or chronically impairing conditions that affect about 6% of the world's population. RDs are often called 'orphan' diseases, since people suffering from them attract little support from national health systems. Aim: The aim of this study is to describe the clinical characteristics of, and the available laboratory examinations for, patients who were hospitalized in a tertiary referral center and finally received a diagnosis associated with a Rare Neurological Disease (RND). Materials and Methods: Patients that were hospitalized in our clinic from 1 January 2014 to 31 March 2022 and were finally diagnosed with an RND were consecutively included. The RND classification was performed according to the ORPHAcode system. Results: A total of 342 out of 11.850 (2.9%) adult patients admitted to our department during this period received a diagnosis associated with an RND. The most common diagnosis (N = 80, 23%) involved an RND presenting with dementia, followed by a motor neuron disease spectrum disorder (N = 64, 18.7%). Family history indicative of an RND was present in only 21 patients (6.1%). Fifty-five (16%) people had previously been misdiagnosed with another neurological condition. The mean time delay between disease onset and diagnosis was 4.24 ± 0.41 years. Conclusions: Our data indicate that a broad spectrum of RNDs may reach a tertiary Neurological Center after a significant delay. Moreover, our data underline the need for a network of reference centers, both at a national and international level, expected to support research on the diagnosis and treatment of RND.

Prevalence, Infectious Characteristics and Genetic Diversity of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus (MRSA) in Two Raw-Meat Processing Establishments in Northern Greece.

In the present study, we investigated the isolation frequency, the genetic diversity, and the infectious characteristics of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) from the incoming meat and the meat products, the environment, and the workers' nasal cavities, in two meat-processing establishments in northern Greece. The isolated S. aureus strains were examined for their resistance to antimicrobials, carriage of the mecA and mecC genes, carriage of genes encoding for the production of nine staphylococcal enterotoxins, carriage of the Panton-Valentine Leukocidin and Toxic Shock Syndrome genes, and the ability to form biofilm. The genetic diversity of the isolates was evaluated using Pulsed Field Gel Electrophoresis (PFGE) and spa typing. S. aureus was isolated from 13.8% of the 160 samples examined, while only one sample (0.6%) was contaminated by MRSA carrying the mecA gene. The evaluation of the antimicrobial susceptibility of the isolates revealed low antimicrobial resistance. The higher resistance frequencies were observed for penicillin (68.2%), amoxicillin/clavulanic acid (36.4%) and tetracycline (18.2%), while 31.8% of the isolates were sensitive to all antimicrobials examined. Multidrug resistance was observed in two isolates. None of the isolates carried the mecC or lukF-PV genes, and two isolates (9.1%) harbored the tst gene. Eight isolates (36.4%) carried the seb gene, one carried the sed gene, two (9.1%) carried both the sed and sei genes, and one isolate (4.5%) carried the seb, sed and sei genes. Twenty-one (95.5%) of the isolates showed moderate biofilm production ability, while only one (4.5%) was characterized as a strong biofilm producer. Genotyping of the isolates by PFGE indicates that S. aureus from different meat-processing establishments represent separate genetic populations. Ten different spa types were identified, while no common spa type isolates were detected within the two plants. Overall, our findings emphasize the need for the strict application of good hygienic practices at the plant level to control the spread of S. aureus and MRSA to the community through the end products.

Ocrelizumab in Patients with Active Primary Progressive Multiple Sclerosis: Clinical Outcomes and Immune Markers of Treatment Response.

Ocrelizumab is a B-cell-depleting monoclonal antibody approved for the treatment of relapsing-remitting multiple sclerosis (RRMS) and active primary progressive MS (aPPMS). This prospective, uncontrolled, open-label, observational study aimed to assess the efficacy of ocrelizumab in patients with aPPMS and to dissect the clinical, radiological and laboratory attributes of treatment response. In total, 22 patients with aPPMS followed for 24 months were included. The primary efficacy outcome was the proportion of patients with optimal response at 24 months, defined as patients free of relapses, free of confirmed disability accumulation (CDA) and free of T1 Gd-enhancing lesions and new/enlarging T2 lesions on the brain and cervical MRI. In total, 14 (63.6%) patients and 13 patients (59.1%) were classified as responders at 12 and 24 months, respectively. Time exhibited a significant effect on mean absolute and normalized gray matter cerebellar volume (F = 4.342, p = 0.23 and F = 4.279, p = 0.024, respectively). Responders at 24 months exhibited reduced peripheral blood ((%) of CD19+ cells) plasmablasts compared to non-responders at the 6-month point estimate (7.69 ± 4.4 vs. 22.66 ± 7.19, respectively, p = 0.043). Response to ocrelizumab was linked to lower total and gray matter cerebellar volume loss over time. Reduced plasmablast depletion was linked for the first time to sub-optimal response to ocrelizumab in aPPMS.

Long-Term Efficacy Outcomes of Natalizumab vs. Fingolimod in Patients With Highly Active Relapsing-Remitting Multiple Sclerosis: Real-World Data From a Multiple Sclerosis Reference Center.

Background: Natalizumab (NTZ) and fingolimod (FTY) are second-line disease modifying treatments (DMTs) approved for Relapsing - Remitting Multiple Sclerosis (RRMS). Few studies are available on a direct comparison between NTZ and FTY, based on post-marketing experience, with conflicting results and reporting relatively short follow-up period. Aim: We hereby report real-world experience of a MS Center with respect to NTZ vs. FTY comparison in terms of efficacy and safety, referencing long-term follow-up. Methods: We used retrospective data for all patients that received 2nd-line treatment NTZ (since May 2007) or FTY (since September 2011). Primary endpoints were, among others, annual EDSS score (mean change from baseline), time to disability worsening or improvement, Annualized Relapse Rate (ARR) after 12 and 24 months and upon total treatment duration, time to first relapse and time to radiological progression. Results: A total of 138 unmatched patients, 84 treated with NTZ and 54 treated with FTY were included. Following Propensity Score (PS) matching, 31 patients in each group were retained. Mean follow-up period for NTZ- and FTY-treated patients was 4.43 ± 0.29 and 3.59 ± 0.32 years (p = 0.057), respectively. In the matched analysis, time to disability improvement and time to disability worsening was comparable between groups. A higher proportion of patients remained free of relapse under NTZ, compared to FTY (Log Rank test p = 0.021, HR: 0.25, 95% CI: 0.08-0.8), as well as free of MRI activity (Log Rank test p = 0.006, HR: 0.26, 95% CI: 0.08-0.6). Treatment discontinuation due to MRI activity was significantly higher for FTY-treated patients compared to NTZ (Log Rank test p = 0.019, HR: 0.12, 95% CI: 0.05-0.76). Conclusion: Our results indicate toward NTZ superiority with respect to relapse and MRI activity outcomes. The fact that NTZ-treated patients may achieve long-standing clinical and radiological remission points toward the need for long follow-up data.

Staphylococcus aureus Isolated from Ruminants with Mastitis in Northern Greece Dairy Herds: Genetic Relatedness and Phenotypic and Genotypic Characterization.

Staphylococcus aureus is the most common mastitis-related pathogen in dairy cattle, goats, and sheep worldwide. However, the population structure and genomic characteristics of mastitis-associated S. aureus in small ruminants are limited. Furthermore, the genotypic and phenotypic characteristics involved in the pathogenicity of S. aureus have been thoroughly defined, yet their association with the severity of mastitis is not fully established. Here, we performed genotyping by pulsed-field gel electrophoresis (PFGE) and spa analyses to assess the genetic diversity and relatedness of 162 S. aureus strains recovered from clinical mastitis (CM) and subclinical mastitis (SCM) cases from goats, sheep, and bovines. PFGE analysis revealed 108 distinguishable pulsotypes and 3 main clusters that comprised isolates from the three host species, while according to spa typing, 32 different spa types were identified. Genotypic analysis revealed a spreading of genetically related or indistinguishable S. aureus strains among ovine, caprine, and bovine farms of distant geographical regions. In total, 28 different staphylococcal enterotoxin (SE) gene profiles were observed, revealing a diverse range of SE genes among isolates. By evaluating the antimicrobial resistance, we found low phenotypic antimicrobial resistance among all ruminant isolates. We also performed multiple correspondence analysis, which indicated that the presence of the sec gene, biofilm production, and high autoaggregation ability are associated with CM cases.

Listeria monocytogenes induced dysbiosis in snails and rebiosis achieved by administration of the gut commensal Lactobacillus plantarum Sgs14 strain.

Listeria monocytogenes strains were isolated from Cornu aspersum maxima snails from farm units experiencing high mortalities and were characterized by phenotypic, molecular and biochemical criteria. A high heterogeneity was observed in the pulsed-field gel electrophoresis (PFGE) pulsotypes as well as in the virulence (13-100% mortality) among the fifteen L. monocytogenes strains. One strain was characterized as non-virulent while three strains exhibited hypervirulent phenotype. Hypervirulence activity was associated with cell surface properties such as hydrophobicity, autoaggregation and biofilm formation, with increased tolerance to snail's gut barriers such as pedal mucus, gastric mucus, gastric juices, and acidic pH as well as with increased capacity to resist the antibacterial activity of snail haemolymph and modulate immune cell populations and functions such as chemotaxis and phagocytoses. L. monocytogenes dysbiosis was characterized by a clinicopathological phenotype including immobilization of snails' headfoot outside the shell, increased mucus-secreting cells in the intestinal epithelium and feces, alteration of intestinal ridges morphology and excessive increase of haemolymph immune cells and cell death. Rebiosis in L. monocytogenes SN3 strain infected snails was achieved by dietary supplementation of the snail-gut commensal probiotic L. plantarum Sgs14 strain by exhibiting anti-Listeria activity, reducing mortality and clinicopathological manifestations as well as exhibiting immunomodulatory activity.

Reduced expression of L-selectin in T-cells correlates with relative lymphocyte increase in patients with RRMS treated with natalizumab - functional implication towards PML risk.

Objectives: Natalizumab (NTZ), a treatment indicated for patients with highly active Relapsing - Remitting Multiple Sclerosis (RRMS), is known to induce increased relative frequency of lymphocytes. Progressive Multifocal Leukoencephalitis (PML) is a rare but serious adverse event related to NTZ. Moreover, reduced L-selectin (CD62L) expression in T-cells in cryopreserved samples of patients with RRMS under NTZ has been proposed as a biomarker of pre-PML state. We explore the association between L-selectin expression in T-cells and hematological parameters in freshly processed samples of patients with RRMS under NTZ.Methods: We studied L-selectin expression in patients with: RRMS under NTZ (n=34), fingolimod (FTY, n=14), interferon-beta (IFNß, n=22), glatiramer acetate (GA, N=17); in 9 patients with secondary progressive (SP) MS and in 6 healthy controls. Twenty-two patients under NTZ and 6 patients under FTY were followed for 18 months. One NTZ-treated patient developed PML during the study.Results: Patients under NTZ exhibited increased relative frequency of lymphocytes (40.02±1.45) compared to patients under first-line treatment (30.57±1.68, p<0.001) and to patients with SPMS (29±1.56, p=0.02), and a lower mean L-selectin expression in (69.39±1.73) compared to patients under first-line treatment (79.1±1.17, p=0.003). A negative correlation between the relative frequency of CD4+CD62L+ T-cells and the absolute lymphocyte counts (Pearson's r=0.367, p=0.033) was observed.Discussion: We hereby provide mechanistic insight in a possible pathway implicated in NTZ-related PML risk. These results further underline the need for thorough validation of L-selectin expression in T-cells as a potential pre-PML biomarker.

Estimating Everyday Neuropsychological Functioning in Multiple Sclerosis: Reliability and Validity of the Greek Multiple Sclerosis Neuropsychological Questionnaire.

The Multiple Sclerosis Neuropsychological Questionnaire is a brief screening questionnaire for the assessment of everyday neuropsychological competence of patients with Multiple Sclerosis. The aim of the present study was to examine psychometric properties of the Greek version of the instrument. One hundred and three MS patients and 60 informants participated in the present study and completed the questionnaire. From the initial patient sample, 51 participants completed broadly used neuropsychological tests and measures estimating cognitive failures and depression. Moreover, after a six-month interval the MSNQ was administered to 58 patients from the initial sample in order to explore test-retest reliability. Cronbach's α was 0.92 and 0.93 for patient and informant forms, respectively. The patient form was correlated significantly with measures of cognitive failures and depression. Low correlations were found between the informant form and performance on cognitive tests. In regard to the patient form, significant correlation was observed between repeated administrations and, psychometrically, the three-factor structure was preferable than the one-factor structure. The present study confirms the already established pattern of correlations among the two MSNQ forms, neuropsychological test performance and depression measurements. Additional research is needed in order to define a cut-off score for the MSNQ-I providing further information about the diagnostic interpretability of the instrument.

The Impact of Methylprednisolone Pulses during Relapses of Multiple Sclerosis on the Kinetics of Anti-Interferon-Beta Antibodies.

BACKGROUND/AIMS: We assessed the, hitherto unknown, impact of intravenous methylprednisolone (ivMP) pulses during relapses of multiple sclerosis (MS) on the kinetics of anti-interferon-beta neutralizing antibodies (Nabs) and binding antibodies (Babs). METHODS: Babs (ELISA) and Nabs (antiviral cytopathic effect assay) titers were evaluated before, immediately after and at 1 month following ivMP in 60 MS patients. RESULTS: ivMP reduces Nabs and Babs titers for at least 1 month. Baseline titers determine Nabs and Babs seronegativity at the end of ivMP. Clinical response to ivMP tends to be better in Nabs(+) patients. CONCLUSION: Sampling for Nabs/Babs should be avoided during or shortly after ivMP to avoid transient positive or negative results that may obscure the decision to switch treatment.