The first description of the complete genome sequence of multidrug-resistant Salmonella enterica serovar monophasic Typhimurium (1,4,[5],12:i:-) isolate with the mcr-1.1 gene on IncHI2 found in pig in Poland.

Monophasic Salmonella Typhimurium (1,4,[5],12:i:-) is one of the leading Salmonella serovars causing human salmonellosis in Europe. It has been observed in Poland since 2008. This serovar is considered the one with the highest rate of mcr prevalence. This report presents a sequence characteristic of the multidrug-resistant (MDR) monophasic S. Typhimurium isolated from a pig faecal sample with the confirmed presence of the mcr-1.1 gene. The genome was assembled into the complete chromosome and 4 plasmids: IncHI2 (232 119 bp), IncFIB/IncFIC (133 901 bp), ColRNAI (6659 bp), and Col8282 (4066bp). The strain identified as ST34 carried multiple antimicrobial resistance genes located both on chromosome (tet(B)) and plasmids: mcr-1.1 and blaTEM-1B on ST4-IncHI2, and mef(B), blaTEM-1B, aadA1, qacL, dfrA12, aadA2, cmlA1, sul3, tet(M) on IncFIB/FIC. The mcr-1.1 gene was previously identified in E. coli deriving mainly from poultry, but this is the first case of the occurrence of mcr-positive Salmonella in Poland. The obtained results of analysis of the genome content draw attention to the problem of multidrug-resistant pathogens, especially in the context of resistance to colistin which is a last-resort antimicrobial.

Cryptic SARS-CoV-2 lineage identified on two mink farms as a possible result of long-term undetected circulation in an unknown animal reservoir, Poland, November 2022 to January 2023.

In late 2022 and early 2023, SARS-CoV-2 infections were detected on three mink farms in Poland situated within a few km from each other. Whole-genome sequencing of the viruses on two of the farms showed that they were related to a virus identified in humans in the same region 2 years before (B.1.1.307 lineage). Many mutations were found, including in the S protein typical of adaptations to the mink host. The origin of the virus remains to be determined.

Attempting to Identify Bacterial Allies in Immunotherapy of NSCLC Patients.

Introduction: Factors other than PD-L1 (Programmed Death Ligand 1) are being sought as predictors for cancer immuno- or chemoimmunotherapy in ongoing studies and long-term observations. Despite high PD-L1 expression on tumor cells, some patients do not benefit from immunotherapy, while others, without the expression of this molecule, respond to immunotherapy. Attention has been paid to the composition of the gut microbiome as a potential predictive factor for immunotherapy effectiveness. Materials and Methods: Our study enrolled 47 Caucasian patients with stage IIIB or IV non-small cell lung cancer (NSCLC). They were eligible for treatment with first- or second-line immunotherapy or chemoimmunotherapy. We collected stool samples before the administration of immunotherapy. We performed next-generation sequencing (NGS) on DNA isolated from the stool sample and analyzed bacterial V3 and V4 of the 16S rRNA gene. Results: We found that bacteria from the families Barnesiellaceae, Ruminococcaceae, Tannerellaceae, and Clostridiaceae could modulate immunotherapy effectiveness. A high abundance of Bacteroidaaceae, Barnesiellaceae, and Tannerellaceae could extend progression-free survival (PFS). Moreover, the risk of death was significantly higher in patients with a high content of Ruminococcaceae family (HR = 6.3, 95% CI: 2.6 to 15.3, p < 0.0001) and in patients with a low abundance of Clostridia UCG-014 (HR = 3.8, 95% CI: 1.5 to 9.8, p = 0.005) regardless of the immunotherapy line. Conclusions: The Clostridia class in gut microbiota could affect the effectiveness of immunotherapy, as well as the length of survival of NSCLC patients who received this method of treatment.

Presence of Akkermansiaceae in gut microbiome and immunotherapy effectiveness in patients with advanced non-small cell lung cancer.

The significance of Akkermansia bacteria presence in gut micobiome, mainly Akkermansia mucinifila, is currently being investigated in the context of supporting therapy and marker for response to immunotherapy in cancer patients. It is indicated that patients with non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors (ICIs) respond better to treatment if this bacterium is present in the intestine.We performed next-generation sequencing of the gut microbiome from patients treated in the first or second line therapy with anti-PD-1 (anti-programmed death 1) or anti-PD-L1 (anti-programmed death ligand 1) monoclonal antibodies. In our study group of 47 NSCLC patients, the percentage of Akkermansiaceae was higher in patients with disease stabilization and with partial response to immunotherapy compared to patients with disease progression. Moreover, we found that a higher percentage of Akkermansiaceae was present in patients with squamous cell carcinoma compared to adenocarcinoma. Our study showed that Akkermansiaceae could be supporting marker for response to immunotherapies in NSCLC patients, nonetheless further in-depth studies should be conducted in the role of Akkermansiaceae in cancer immunotherapy.

SARS-CoV-2 Monitoring on Mink Farms in Poland.

Introduction: Many countries have reported severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infections in mink, and transmission back to humans has raised the concern of novel variants emerging in these animals. The monitoring system on Polish mink farms detected SARS-CoV-2 infection first in January 2021 and has been kept in place since then. Material and Methods: Oral swab samples collected between February 2021 and March 2022 from 11,853 mink from 594 farms in different regions of Poland were screened molecularly for SARS-CoV-2. Isolates from those with the highest loads of viral genetic material from positive farms were sequenced and phylogenetically analysed. Serological studies were also carried out for one positive farm in order to follow the antibody response after infection. Results: SARS-CoV-2 RNA was detected in mink on 11 farms in 8 out of 16 Polish administrative regions. Whole genome sequences were obtained for 19 SARS-CoV-2 strains from 10 out of 11 positive farms. These genomes belonged to four different variants of concern (VOC) - VOC-Gamma (20B), VOC-Delta (21J), VOC-Alpha (20I) and VOC-Omicron (21L) - and seven different Pango lineages - B.1.1.464, B.1.1.7, AY.43, AY.122, AY.126, B.1.617.2 and BA.2. One of the nucleotide and amino acid mutations specific for persistent strains found in the analysed samples was the Y453F host adaptation mutation. Serological testing of blood samples revealed a high rate of seroprevalence on the single mink farm studied. Conclusion: Farmed mink are highly susceptible to infection with SARS-CoV-2 of different lineages, including Omicron BA.2 VOC. As these infections were asymptomatic, mink may become an unnoticeable virus reservoir generating new variants potentially threatening human health. Therefore, real-time monitoring of mink is extremely important in the context of the One Health approach.

Mink SARS-CoV-2 Infection in Poland - Short Communication.

INTRODUCTION: Since April 2020, when the first SARS-CoV-2 infection was reported in mink and subsequently in mink farm workers in the Netherlands, it has been confirmed that human-to-mink and mink-to-human transmission can occur. Later, SARS-CoV-2 infections in mink were reported in many European and North American countries. MATERIAL AND METHODS: Samples from 590 mink from a total of 28 farms were tested by real-time RT-PCR. Whole genome sequences from one positive farm were generated and genetic relatedness was established. RESULTS: SARS-CoV-2 RNA was detected on a breeder farm with stock of 5,850 mink. Active viraemia was confirmed in individually tested samples with Ct values respectively between 19.4 and 29.6 for E and N gene fragments. Further testing of samples from culled animals revealed 70% positivity in throat swabs and 30% seropositivity in blood samples. Phylogenetic analysis of full-length nucleotide sequences of two SARS-CoV-2 isolates revealed that they belong to the 20B Nextstrain clade. Several nucleotide mutations were found in analysed samples compared to the reference Wuhan HU-1 strain and some of them were nonsynonymous. CONCLUSION: We report the infection of mink with SARS-CoV-2 on one farm in Poland and the results of subsequent analysis of virus sequences from two isolates. These data can be useful for assessment of the epidemiological situation of SARS-CoV-2 in Poland and how it endangers public health.

Whole-genome sequence analysis of Salmonella Infantis isolated from raw chicken meat samples and insights into pESI-like megaplasmid.

There has been an increase in the number of reports on Salmonella enterica subsp. enterica serovar Infantis (S. Infantis) isolated from animals and humans. Recent studies using whole genome sequencing (WGS) have provided evidence on the likely contribution of a unique conjugative megaplasmid (pESI; ~280 kb) to the dissemination of this serovar worldwide. In the present study, twenty-two unrelated Salmonella strains [S. Infantis (n = 20) and Salmonella 6,7:r:- (n = 2)] and their plasmids were investigated using next generation sequencing technologies (MiSeq and MinION) to unravel the significant expansion of this bacteria in Turkey. Multi-locus sequence typing, plasmid replicons, resistance gene contents as well as phylogenetic relations between strains were determined. According to the WGS data, all S. Infantis possessed the relevant megaplasmid backbone genes and belonged to sequence type 32 (ST32) with the exception of a single novel ST7091. Tetracycline and trimethoprim/sulfamethoxazole resistance were found to be widespread in S. Infantis strains and the resistant strains exclusively carried the tetA, sul1, sul2 and dfrA14 genes. One S. Infantis isolate was also a carrier of the plasmid-mediated ampC via blaCMY-2, gene. Moreover, full genomes of four S. Infantis isolates were reconstructed based on hybrid assembly. All four strains contained large plasmids (240-290 kb) similar to previously published megaplasmid (pESI) and accompanied by several small plasmids. The megaplasmid backbone contained a toxin-antitoxin system, two virulence cassettes and segments associated with heavy metals resistance, while variable regions possessed several antibiotic resistance genes flanked by mobile elements. This study indicated that pESI-like megaplasmid is widely disseminated within the tested S. Infantis strains of chicken meat, warranting further genomic studies on clinical strains from humans and animals to uncover the overall emergence and spread of this serovar.

A Turkey-origin H9N2 Avian Influenza Virus Shows Low Pathogenicity but Different Within-Host Diversity in Experimentally Infected Turkeys, Quail and Ducks.

Avian influenza virus (AIV) is a highly diverse and widespread poultry pathogen. Itsevolution and adaptation may be affected by multiple host and ecological factors, which are stillpoorly understood. In the present study, a turkey-origin H9N2 AIV was used as a model toinvestigate the within-host diversity of the virus in turkeys, quail and ducks in conjunction with theclinical course, shedding and seroconversion. Ten birds were inoculated oculonasally with a doseof 106 EID50 of the virus and monitored for 14 days. Virus shedding, transmission andseroconversion were evaluated, and swabs collected at selected time-points were characterized indeep sequencing to assess virus diversity. In general, the virus showed low pathogenicity for theexamined bird species, but differences in shedding patterns, seroconversion and clinical outcomewere noted. The highest heterogeneity of the virus population as measured by the number of singlenucleotide polymorphisms and Shannon entropy was found in oropharyngeal swabs from quail,followed by turkeys and ducks. This suggests a strong bottleneck was imposed on the virus duringreplication in ducks, which can be explained by its poor adaptation and stronger selection pressurein waterfowl. The high within-host virus diversity in quail with high level of respiratory sheddingand asymptomatic course of infection may contribute to our understanding of the role of quail asan intermediate host for adaptation of AIV to other species of poultry. In contrast, low viruscomplexity was observed in cloacal swabs, mainly from turkeys, showing that the within-hostdiversity may vary between different replication sites. Consequences of these observations on thevirus evolution and adaptation require further investigation.

Evaluation of Direct Metagenomics and Target Enriched Approaches for High-throughput Sequencing of Field Rabies Viruses.

INTRODUCTION: High-throughput sequencing (HTS) identifies random viral fragments in environmental samples metagenomically. High reliability gains it broad application in virus evolution, host-virus interaction, and pathogenicity studies. Deep sequencing of field samples with content of host genetic material and bacteria often produces insufficient data for metagenomics and must be preceded by target enrichment. The main goal of the study was the evaluation of HTS for complete genome sequencing of field-case rabies viruses (RABVs). MATERIAL AND METHODS: The material was 23 RABVs isolated mainly from red foxes and one European bat lyssavirus-1 isolate propagated in neuroblastoma cells. Three methods of RNA isolation were tested for the direct metagenomics and RABV-enriched approaches. Deep sequencing was performed with a MiSeq sequencer (Illumina) and reagent v3 kit. Bioinformatics data were evaluated by Kraken and Centrifuge software and de novo assembly was done with metaSPAdes. RESULTS: Testing RNA extraction procedures revealed the deep sequencing scope superiority of the combined TRIzol/column method. This HTS methodology made it possible to obtain complete genomes of all the RABV isolates collected in the field. Significantly greater rates of RABV genome coverages (over 5,900) were obtained with RABV enrichment. Direct metagenomic studies sequenced the full length of 6 out of 16 RABV isolates with a medium coverage between 1 and 71. CONCLUSION: Direct metagenomics gives the most realistic illustration of the field sample microbiome, but with low coverage. For deep characterisation of viruses, e.g. for spatial and temporal phylogeography during outbreaks, target enrichment is recommended as it covers sequences much more completely.

Analysis of the immune response of calves to various saponin-based adjuvants for an experimental Mycoplasma bovis vaccine.

Mycoplasma bovis is a primary infectious agent of many disorders in cattle including bovine respiratory disease. No commercial vaccines against M. bovis are available in Europe. The immune response of calves to three saponin-based adjuvants combined with a field Polish M. bovis strain was evaluated. Four groups of six calves each were injected subcutaneously with the M. bovis strain combined with either saponin, saponin + Emulsigen®, saponin + Emulsigen® + alphatocopherol acetate, or with phosphate-buffered saline as control group. Blood and nasal swab samples were collected up to day 84 post injection. All formulations effectively stimulated the humoral and the cellular immune response of the calves, but the course of the response depended on the adjuvant formulation. These immunological data provide additional information supporting the findings of previous M. bovis saponin and Emulsigen® vaccine challenge studies to facilitate the development of successful M. bovis vaccines.

[Neurofeedback therapy influence on clinical status and some EEG parameters in children with localized epilepsy]. / Wplyw terapii neurofeedback na stan kliniczny i wybrane parametry zapisu EEG u dzieci chorych na padaczke z napadami czesciowymi.

UNLABELLED: Neurofeedback (EEG biofeedback, NF) is one of the methods of non-pharmacological instrumental therapy. In the treatment of epilepsy it is considered as a complementary method, reducing the number of seizures. The aim of this study was to investigate the effect of NF on the incidence of clinical attacks and bioelectrical activity of the brain in children with epilepsy with partial seizures and secondarily generalized seizures. MATERIAL AND METHODS: The study involved a group of 78 children with partial seizures, additionally 30 of them had second-ry generalized seizures. There were 35 girls and 43 boys ranging in age from 5 to 17.5 years. They were treated with NF in the Neuromedica Institute in Krakow, in the period 2005-2015. Treatment time ranged from 6 months to 3 years. We have evaluated the incidence of seizures before and after NF therapy. Visual analysis of EEG recordings with assessment of background activity, location of focal changes, presence of paroxysmal pattern and epileptic graphoelements. RESULTS: In 41/78 children seizures occurred 1-2 times a month, in 19/78 every day and 9 children were diagnosed with epileptic syndrome. During NF therapy 52/78 children experienced resolution of clinical seizures. NF therapy was ineffective in only one child. In 18 child withdrawal of antiepileptic treatment was achieved and in 22 reduction of drug doses. Epileptic graphoelements occurred in 46/78 children before treatment and in 21/78 children after treatment. Before NF therapy irregular bio-electrical activity was found in 51 children versus 27 children with regular bioelectric activity and in 27 children versus 51 children after the therapy. The amplitude of this activity returned to normal in 19 children, and in 29 children the frequency of background activity. Particularly surprising was to restore synchrony and symmetry of EEG pattern in 44 children (11 children before NF treatment to 55 children after the treatment). Even more significant effect of NF therapy was observed in EEG patterns, abolition of paroxysmal pattern and reduction of continuous, localized and generalized changes. NF therapy also advantageously reduced the number of localized changes and decreased sensitivity of EEG pattern to hyperventilation and photostimulation. CONCLUSIONS: During NF therapy in almost all children with epileptic partial seizures and secondarily generalized seizures resolution or reduction of clinical seizures was observed. In nearly 1/4 of children withdrawal of antiepileptic treatment was achieved and in higher number reduction of pharmacotherapy. All parameters of the background EEG bioelectric activity in these children have improved, especially synchrony and symmetry of EEG pattern and paroxysmal changes subsided.