Understanding Vietnamese chicken farmers' knowledge and practices related to antimicrobial resistance using an item response theory approach.

Introduction: Antimicrobial resistance (AMR) poses a threat to animal and human health, as well as food security and nutrition. Development of AMR is accelerated by over- and misuse of antimicrobials as seen in many livestock systems, including poultry production. In Vietnam, high AMR levels have been reported previously within poultry production, a sector which is dominated by small-scale farming, even though it is intensifying. This study focuses on understanding small- and medium-scale chicken farmers' knowledge and practices related to AMR by applying an item response theory (IRT) approach, which has several advantages over simpler statistical methods. Methods: Farmers representing 305 farms in Thai Nguyen province were interviewed from November 2021 to January 2022, using a structured questionnaire. Results generated with IRT were used in regression models to find associations between farm characteristics, and knowledge and practice levels. Results: Descriptive results showed that almost all farmers could buy veterinary drugs without prescription in the local community, that only one third of the farmers received veterinary professional advice or services, and that the majority of farmers gave antibiotics as a disease preventive measure. Regression analysis showed that multiple farm characteristics were significantly associated to farmers' knowledge and practice scores. Conclusion: The study highlights the complexity when tailoring interventions to move towards more medically rational antibiotic use at farms in a setting with high access to over-the-counter veterinary drugs and low access to veterinary services, since many on-farm factors relevant for the specific context need to be considered.

Tumor Predisposing Post-Zygotic Chromosomal Alterations in Bladder Cancer-Insights from Histologically Normal Urothelium.

Bladder urothelial carcinoma (BLCA) is the 10th most common cancer with a low survival rate and strong male bias. We studied the field cancerization in BLCA using multi-sample- and multi-tissue-per-patient protocol for sensitive detection of autosomal post-zygotic chromosomal alterations and loss of chromosome Y (LOY). We analysed 277 samples of histologically normal urothelium, 145 tumors and 63 blood samples from 52 males and 15 females, using the in-house adapted Mosaic Chromosomal Alterations (MoChA) pipeline. This approach allows identification of the early aberrations in urothelium from BLCA patients. Overall, 45% of patients exhibited at least one alteration in at least one normal urothelium sample. Recurrence analysis resulted in 16 hotspots composed of either gains and copy number neutral loss of heterozygosity (CN-LOH) or deletions and CN-LOH, encompassing well-known and new BLCA cancer driver genes. Conservative assessment of LOY showed 29%, 27% and 18% of LOY-cells in tumors, blood and normal urothelium, respectively. We provide a proof of principle that our approach can characterize the earliest alterations preconditioning normal urothelium to BLCA development. Frequent LOY in blood and urothelium-derived tissues suggest its involvement in BLCA.

Expression of influenza A virus glycan receptor candidates in mallard, chicken, and tufted duck.

Influenza A virus (IAV) pandemics result from interspecies transmission events within the avian reservoir and further into mammals including humans. Receptor incompatibility due to differently expressed glycan structures between species has been suggested to limit zoonotic IAV transmission from the wild bird reservoir as well as between different bird species. Using glycoproteomics, we have studied the repertoires of expressed glycan structures with focus on putative sialic acid-containing glycan receptors for IAV in mallard, chicken and tufted duck; three bird species with different roles in the zoonotic ecology of IAV. The methodology used pinpoints specific glycan structures to specific glycosylation sites of identified glycoproteins and was also used to successfully discriminate α2-3- from α2-6-linked terminal sialic acids by careful analysis of oxonium ions released from glycopeptides in tandem MS/MS (MS2), and MS/MS/MS (MS3). Our analysis clearly demonstrated that all three bird species can produce complex N-glycans including α2-3-linked sialyl Lewis structures, as well as both N- and O- glycans terminated with both α2-3- and α2-6-linked Neu5Ac. We also found the recently identified putative IAV receptor structures, Man-6P N-glycopeptides, in all tissues of the three bird species. Furthermore, we found many similarities in the repertoires of expressed receptors both between the bird species investigated and to previously published data from pigs and humans. Our findings of sialylated glycan structures, previously anticipated to be mammalian specific, in all three bird species may have major implications for our understanding of the role of receptor incompatibility in interspecies transmission of IAV.

Early Staphylococcal Periprosthetic Joint Infection (PJI) Treated with Debridement, Antibiotics, and Implant Retention (DAIR): Inferior Outcomes in Patients with Staphylococci Resistant to Rifampicin.

It is unknown how rifampicin resistance in staphylococci causing a periprosthetic joint infection (PJI) affects outcomes after debridement, antibiotics, and implant retention (DAIR). We thus aimed to compare the risk of relapse in DAIR-treated early PJI caused by staphylococci with or without rifampicin resistance. In total, 81 patients affected by early PJI were included, and all patients were treated surgically with DAIR. This was repeated if needed. The endpoint of relapse-free survival was estimated using the Kaplan-Meier method, and Cox regression models were fitted to assess the risk of infection relapse for patients infected with rifampicin-resistant bacteria, adjusted for age, sex, type of joint, and type of index surgery. In patients with rifampicin-resistant staphylococci, relapse was seen in 80% after one DAIR procedure and in 70% after two DAIR procedures. In patients with rifampicin-sensitive bacteria, 51% had an infection relapse after one DAIR procedure and 33% had an infection relapse after two DAIR procedures. Patients with rifampicin-resistant staphylococcal PJI thus had an increased adjusted risk of infection relapse of 1.9 (95% CI: 1.1-3.6, p = 0.04) after one DAIR procedure compared to patients with rifampicin-sensitive bacteria and a 4.1-fold (95% CI: 1.2-14.1, p = 0.03) increase in risk of infection relapse after two DAIR procedures. Staphylococcal resistance to rifampicin is associated with inferior outcomes after DAIR. These findings suggest that DAIR may not be a useful strategy in early PJI caused by rifampicin-resistant staphylococci.

Incidence of Rifampicin Resistance in Periprosthetic Joint Infection: A Single-Centre Cohort Study on 238 Patients.

BACKGROUND: Rifampicin is a pillar in the treatment of periprosthetic joint infection (PJI). However, rifampicin resistance is an increasing threat to PJI treatment. This study explores the incidence of rifampicin-resistant bacteria over time in a Swedish tertiary referral centre and the association of rifampicin resistance with infection-free survival after PJI. METHODS: The study included 238 staphylococcal PJIs treated between 2001 and 2020 for which susceptibility data for rifampicin were available. Data on causative bacteria, rifampicin resistance, treatment, and outcome were obtained. Kaplan-Meier survival analysis and Cox regression modelling estimated the infection-free cumulative survival and adjusted hazard ratios (HRs) for the risk of treatment failure. RESULTS: Rifampicin-resistant causative bacteria were identified in 40 cases (17%). The proportion of rifampicin-resistant agents decreased from 24% in 2010-2015 to 12% in 2016-2020. The 2-year infection-free survival rates were 78.6% (95% CI, 66.4-93.1%) for the rifampicin-resistant group and 90.0% (95% CI, 85.8-94.4%) for the rifampicin-sensitive group. Patients with PJI caused by rifampicin-resistant bacteria had an increased risk of treatment failure (adjusted HR, 4.2; 95% CI, 1.7-10.3). CONCLUSIONS: The incidence of PJI caused by rifampicin-resistant bacteria did not increase over the past 20 years. The risk of treatment failure in PJI caused by rifampicin-resistant bacteria is more than four times that caused by rifampicin-sensitive bacteria, highlighting the importance of limiting the development of rifampicin resistance.

Temporal pathway analysis of cerebrospinal fluid proteome in herpes simplex encephalitis.

OBJECTIVES: We examined the temporal changes of the CSF proteome in patients with herpes simplex encephalitis (HSE) during the course of the disease, in relation to anti-N-methyl-D-aspartate receptor (NMDAR) serostatus, corticosteroid treatment, brain MRI and neurocognitive performance. METHODS: Patients were retrospectively included from a previous prospective trial with a pre-specified CSF sampling protocol. Mass spectrometry data of the CSF proteome were processed using pathway analysis. RESULTS: We included 48 patients (110 CSF samples). Samples were grouped based on time of collection relative to hospital admission - T1: ≤ 9 d, T2: 13-28 d, T3: ≥ 68 d. At T1, a strong multi-pathway response was seen including acute phase response, antimicrobial pattern recognition, glycolysis and gluconeogenesis. At T2, most pathways activated at T1 were no longer significantly different from T3. After correction for multiplicity and considering the effect size threshold, 6 proteins were significantly less abundant in anti-NMDAR seropositive patients compared to seronegative: procathepsin H, heparin cofactor 2, complement factor I, protein AMBP, apolipoprotein A1 and polymeric immunoglobulin receptor. No significant differences in individual protein levels were found in relation to corticosteroid treatment, size of brain MRI lesion or neurocognitive performance. CONCLUSIONS: We show a temporal change in the CSF proteome in HSE patients during the course of the disease. This study provides insight into quantitative and qualitative aspects of the dynamic pathophysiology and pathway activation patterns in HSE and prompts for future studies on the role of apolipoprotein A1 in HSE, which has previously been associated with NMDAR encephalitis.

Reduced Binding between Omicron B.1.1.529 and the Human ACE2 Receptor in a Surrogate Virus Neutralization Test for SARS-CoV-2.

The current gold standard assay for detecting neutralizing antibodies (NAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the conventional virus neutralization test (cVNT), which requires infectious virus and a biosafety level 3 laboratory. Here, we report the development of a SARS-CoV-2 surrogate virus neutralization test (sVNT) that, with Luminex technology, detects NAbs. The assay was designed to mimic the virus-host interaction and is based on antibody blockage between the human angiotensin-converting enzyme 2 (hACE2) receptor and the spike (S) protein of the Wuhan, Delta, and Omicron (B.1.1.529) variants of SARS-CoV-2. The sVNT proved to have a 100% correlation with a SARS-CoV-2 cVNT regarding qualitative results. Binding between the hACE2 receptor and the S1 domain of the B.1.1.529 lineage of the Omicron variant was not observed in the assay but between the receptor and an S1 + S2 trimer and the receptor binding domain (RBD) in a reduced manner, suggesting less efficient receptor binding for the B.1.1.529 Omicron variant. The results indicate that the SARS-CoV-2 sVNT is a suitable tool for both the research community and the public health service, as it may serve as an efficient diagnostic alternative to the cVNT.

A Comparison of Host Responses to Infection with Wild-Type Avian Influenza Viruses in Chickens and Tufted Ducks.

Cross-species transmission of influenza A virus (IAV) from wild waterfowl to poultry is the first step in a chain of events that can ultimately lead to exposure and infection of humans. Herein, we study the outcome of infection with eight different mallard-origin IAV subtypes in two different avian hosts: tufted ducks and chickens. We found that infection and shedding patterns as well as innate immune responses were highly dependent on viral subtypes, host species, and inoculation routes. For example, intraoesophageal inoculation, commonly used in mallard infection experiments, resulted in no infections in contrast to oculonasal inoculation, suggesting a difference in transmission routes. Despite H9N2 being endemic in chickens, inoculation of mallard-origin H9N2 failed to cause viable infection beyond 1 day postinfection in our study design. The innate immune responses were markedly different in chickens and tufted ducks, and despite the presence of retinoic acid-inducible gene-I (RIG-I) in tufted duck transcriptomes, it was neither up nor downregulated in response to infection. Overall, we have revealed the heterogeneity of infection patterns and responses in two markedly different avian hosts following a challenge with mallard-origin IAV. These virus-host interactions provide new insights into important aspects of interspecies transmission of IAV. IMPORTANCE Our current findings highlight important aspects of IAV infection in birds that have implications for our understanding of its zoonotic ecology. In contrast to mallards where the intestinal tract is the main site of IAV replication, chickens and tufted ducks show limited or no signs of intestinal infection suggesting that the fecal-oral transmission route might not apply to all bird IAV host species. Our results indicate that mallard-origin IAVs undergo genetic changes upon introduction into new hosts, suggesting rapid adaptation to a new environment. However, similar to the mallard, chickens and tufted ducks show a limited immune response to infection with low pathogenic avian influenza viruses. These findings and future studies in different IAV hosts are important for our understanding of barriers to IAV transmission between species and ultimately from the wild reservoir to humans.

Exploring the epidemiology of mcr genes, genetic context and plasmids in Enterobacteriaceae originating from pigs and humans on farms in Thailand.

OBJECTIVES: In veterinary medicine, colistin has been widely used as therapeutic and prophylactic agent, and for growth promotion. However, colistin has been re-introduced into treatment of human MDR bacterial infections. We assessed the characteristics and spread of plasmid-borne colistin resistance among healthy pigs, workers with animal-contact and their household members in Thailand. METHODS: WGS and MIC data of 146 mcr-positive isolates from a cross-sectional One Health study were analysed. Long-read sequencing and conjugation were performed for selected isolates. RESULTS: mcr-carrying isolates were detected in 38% of pooled-pig samples and 16% of human faecal samples. Of 143 Escherichia coli and three Escherichia fergusonii, mcr-1, mcr-3, and mcr-9 variants were identified in 96 (65.8%), 61 (41.8%) and one (0.7%) isolate, respectively. Twelve E. coli co-harboured two mcr variants (mcr-1 and mcr-3). Clonal transmission was detected in five out of 164 farms. mcr-1 was mostly harboured by epidemic IncX4 and IncHI1 plasmids (89.9%). Conversely, mcr-3 was harboured by a range of different plasmids. Comparative plasmid studies suggested IncP and IncFII plasmids as possible endemic mcr-3 plasmids in Asian countries. Moreover, mcr-3 was associated with different mobile genetic elements including TnAs2, ISKpn40 and IS26/15DI. Detected genetic signatures (DRs) indicated recent mcr-3 transpositions, underlining the mobilizable nature of the mcr-3 cassette. CONCLUSIONS: The epidemiology of mcr and the possible evolution of successful plasmids and transposition modules should be carefully monitored. Of special concern is the growing number of different horizontal gene transferring pathways encompassing various transposable modules the mcr genes can be shared between bacteria.

An oseltamivir-resistant avian H1N1 influenza A virus can transmit from mallards to chickens similarly to a wild-type strain: implications for the risk of resistance transmission to humans.

The neuraminidase inhibitor (NAI) oseltamivir is stockpiled globally as part of influenza pandemic preparedness. However, oseltamivir carboxylate (OC) resistance develops in avian influenza virus (AIV) infecting mallards exposed to environmental-like OC concentrations, suggesting that environmental resistance is a real concern. Herein we used an in vivo model to investigate if avian influenza H1N1 with the OC-resistant mutation NA-H274Y (51833/H274Y) as compared to the wild-type (wt) strain (51833 /wt) could transmit from mallards, which would potentially be exposed to environmentally contaminated environments, to and between chickens, thus posing a potential zoonotic risk of antiviral-resistant AIV. Regardless of whether the virus had the OC-resistant mutation or not, chickens became infected both through experimental infection, and following exposure to infected mallards. We found similar infection patterns between 51833/wt and 51833/H274Y such that, one chicken inoculated with 51833/wt and three chickens inoculated with 51833/H274Y were AIV positive in oropharyngeal samples more than 2 days consecutively, indicating true infection, and one contact chicken exposed to infected mallards was AIV positive in faecal samples for 3 consecutive days (51833/wt) and another contact chicken for 4 consecutive days (51833/H274Y). Importantly, all positive samples from chickens infected with 51833/H274Y retained the NA-H274Y mutation. However, none of the virus strains established sustained transmission in chickens, likely due to insufficient adaptation to the chicken host. Our results demonstrate that an OC-resistant avian influenza virus can transmit from mallards and replicate in chickens. NA-H274Y does not constitute a barrier to interspecies transmission per se, as the resistant virus did not show reduced replicative capacity compared to the wild-type counterpart. Thus, responsible use of oseltamivir and surveillance for resistance development is warranted to limit the risk of an OC-resistant pandemic strain.

Antibiotic use during coronavirus disease 2019 intensive care unit shape multidrug resistance bacteriuria: A Swedish longitudinal prospective study.

Objectives: High frequency of antimicrobial prescription and the nature of prolonged illness in COVID-19 increases risk for complicated bacteriuria and antibiotic resistance. We investigated risk factors for bacteriuria in the ICU and the correlation between antibiotic treatment and persistent bacteria. Methods: We conducted a prospective longitudinal study with urine from indwelling catheters of 101 ICU patients from Uppsala University Hospital, Sweden. Samples were screened and isolates confirmed with MALDI-TOF and whole genome sequencing. Isolates were analyzed for AMR using broth microdilution. Clinical data were assessed for correlation with bacteriuria. Results: Length of stay linearly correlated with bacteriuria (R2 = 0.99, p ≤ 0.0001). 90% of patients received antibiotics, primarily the beta-lactams (76%) cefotaxime, piperacillin-tazobactam, and meropenem. We found high prevalence of Enterococcus (42%) being associated with increased cefotaxime prescription. Antibiotic-susceptible E. coli were found to cause bacteriuria despite concurrent antibiotic treatment when found in co-culture with Enterococcus. Conclusion: Longer stays in ICUs increase the risk for bacteriuria in a predictable manner. Likely, high use of cefotaxime drives Enterococcus prevalence, which in turn permit co-colonizing Gram-negative bacteria. Our results suggest biofilms in urinary catheters as a reservoir of pathogenic bacteria with the potential to develop and disseminate AMR.

Loss of Y in leukocytes as a risk factor for critical COVID-19 in men.

BACKGROUND: The COVID-19 pandemic, which has a prominent social and economic impact worldwide, shows a largely unexplained male bias for the severity and mortality of the disease. Loss of chromosome Y (LOY) is a risk factor candidate in COVID-19 due to its prior association with many chronic age-related diseases, and its impact on immune gene transcription. METHODS: Publicly available scRNA-seq data of PBMC samples derived from male patients critically ill with COVID-19 were reanalyzed, and LOY status was added to the annotated cells. We further studied LOY in whole blood for 211 COVID-19 patients treated at intensive care units (ICU) from the first and second waves of the pandemic. Of these, 139 patients were subject to cell sorting for LOY analysis in granulocytes, low-density neutrophils (LDNs), monocytes, and PBMCs. RESULTS: Reanalysis of available scRNA-seq data revealed LDNs and monocytes as the cell types most affected by LOY. Subsequently, DNA analysis indicated that 46%, 32%, and 29% of critically ill patients showed LOY above 5% cut-off in LDNs, granulocytes, and monocytes, respectively. Hence, the myeloid lineage that is crucial for the development of severe COVID-19 phenotype is affected by LOY. Moreover, LOY correlated with increasing WHO score (median difference 1.59%, 95% HDI 0.46% to 2.71%, p=0.025), death during ICU treatment (median difference 1.46%, 95% HDI 0.47% to 2.43%, p=0.0036), and history of vessel disease (median difference 2.16%, 95% HDI 0.74% to 3.7%, p=0.004), among other variables. In 16 recovered patients, sampled during ICU stay and 93-143 days later, LOY decreased significantly in whole blood and PBMCs. Furthermore, the number of LDNs at the recovery stage decreased dramatically (median difference 76.4 per 10,000 cell sorting events, 95% HDI 55.5 to 104, p=6e-11). CONCLUSIONS: We present a link between LOY and an acute, life-threatening infectious disease. Furthermore, this study highlights LOY as the most prominent clonal mutation affecting the myeloid cell lineage during emergency myelopoiesis. The correlation between LOY level and COVID-19 severity might suggest that this mutation affects the functions of monocytes and neutrophils, which could have consequences for male innate immunity.

Genome-associations of extended-spectrum ß-lactamase producing (ESBL) or AmpC producing E. coli in small and medium pig farms from Khon Kaen province, Thailand.

Thailand is undergoing rapid intensification of livestock production where small subsistence farms and medium sized commercial farms coexist. In medium farms, antimicrobials are prescribed by a veterinarian, whereas in small farms antimicrobial use remains largely unsupervised. The impact of these differences as well as other farming practices on the emergence and composition of antimicrobial resistance genes (ARGs) remains largely unknown. We analyzed 363 genomes of extended-spectrum ß-lactamase producing (ESBL) and/or AmpC producing Escherichia coli recovered from humans and pigs at small and medium farms from the Khon Kaen province, Thailand. We tested for genome-wide associations to identify links between ARGs, host, and farm size. Pig isolates from small farms were associated with mcr and qnr genes conferring resistance to colistin and fluoroquinolones, respectively. In contrast, pig isolates from medium farms were associated with ARGs conferring resistance to drugs commonly used on medium farms (i.e., streptomycin). ESBL plasmids from small farms co-carried ARGs conferring resistance to critically important antimicrobials more frequently compared to plasmid from medium farms. Frequent ARG combinations included blaCTX-M-55 + qnrS1 (29.8% vs 17.5% in small and medium farms, respectively), blaCTX-M-55 + qnrS1 + mcr-3.19 (5% vs 0%), blaCTX-M-14 + qnrS1 (9.3% vs 6.2%), and blaCTX-M-14 + qnrS1 + mcr-1.1 (3.1% vs 0%). The co-location on plasmids of ARGs conferring resistance to critically important antimicrobials as defined by the World Health Organization is concerning, and actions to curb their spread are urgently needed. Legislation on limiting antimicrobial sales and initiatives to better inform farmers and veterinarians on appropriate antimicrobial usage and farm biosecurity could help reduce antimicrobial use on farms.

Trabecular Titanium for Orthopedic Applications: Balancing Antimicrobial with Osteoconductive Properties by Varying Silver Contents.

Periprosthetic joint infection (PJI) and implant loosening are the most common complications after joint replacement surgery. Due to their increased surface area, additively manufactured porous metallic implants provide optimal osseointegration but they are also highly susceptible to bacterial colonization. Antibacterial surface coatings of porous metals that do not inhibit osseointegration are therefore highly desirable. The potential of silver coatings on arthroplasty implants to inhibit PJI has been demonstrated, but the optimal silver content and release kinetics have not yet been defined. A tight control over the silver deposition coatings can help overcome bacterial infections while reducing cytotoxicity to human cells. In this regard, porous titanium sputtered with silver and titanium nitride with increasing silver contents enabled controlling the antibacterial effect against common PJI pathogens while maintaining the metabolic activity of human primary cells. Electron beam melting additively manufactured titanium alloys, coated with increasing silver contents, were physico-chemically characterized and investigated for effects against common PJI pathogens. Silver contents from 7 at % to 18 at % of silver were effective in reducing bacterial growth and biofilm formation. Staphylococcus epidermidis was more susceptible to silver ions than Staphylococcus aureus. Importantly, all silver-coated titanium scaffolds supported primary human osteoblasts proliferation, differentiation, and mineralization up to 28 days. A slight reduction of cell metabolic activity was observed at earlier time points, but no detrimental effects were found at the end of the culture period. Silver release from the silver-coated scaffolds also had no measurable effects on primary osteoblast gene expression since similar expression of genes related to osteogenesis was observed regardless the presence of silver. The investigated silver-coated porous titanium scaffolds may thus enhance osseointegration while reducing the risk of biofilm formation by the most common clinically encountered pathogens.

3D-printed porous Ti6Al4V alloys with silver coating combine osteocompatibility and antimicrobial properties.

Additive manufacturing allows for the production of porous metallic implants for use in orthopaedics, providing excellent mechanical stability and osseointegration. However, the increased surface area of such porous implants also renders them susceptible to bacterial colonization. In this work, two trabecular porous Ti6Al4V alloys produced by electron beam melting were investigated for their osteocompatibility and antimicrobial effects, comparing samples with a silver-coated surface to uncoated samples. Dense grit-blasted Ti samples were used for comparison. The porous samples had pore sizes of 500-600 µm and 5 to 10 µm surface roughness, the silver-coated samples contained 7 at.% Ag, resulting in a cumulative Ag release of 3.5 ppm up to 28 days. Silver reduced the adhesion of Staphylococcus aureus to porous samples and inhibited 72 h biofilm formation by Staphylococcus epidermidis but not that of S. aureus. Primary human osteoblast adhesion, proliferation and differentiation were not impaired in the presence of silver, and expression of osteogenic genes as well as production of mineralized matrix were similar on silver-coated and uncoated samples. Our findings indicate that silver coating of porous titanium implants can achieve antimicrobial effects without compromising osteocompatibility, but higher silver contents may be needed to yield a sustained protection against fast-growing bacteria.

Dissemination of Resistant Escherichia coli Among Wild Birds, Rodents, Flies, and Calves on Dairy Farms.

Antimicrobial resistance (AMR) in bacteria in the livestock is a growing problem, partly due to inappropriate use of antimicrobial drugs. Antimicrobial use (AMU) occurs in Swedish dairy farming but is restricted to the treatment of sick animals based on prescription by a veterinary practitioner. Despite these strict rules, calves shedding antimicrobial resistant Enterobacteriaceae have been recorded both in dairy farms and in slaughterhouses. Yet, not much is known how these bacteria disseminate into the local environment around dairy farms. In this study, we collected samples from four animal sources (fecal samples from calves, birds and rodents, and whole flies) and two environmental sources (cow manure drains and manure pits). From the samples, Escherichia coli was isolated and antimicrobial susceptibility testing performed. A subset of isolates was whole genome sequenced to evaluate relatedness between sources and genomic determinants such as antimicrobial resistance genes (ARGs) and the presence of plasmids were assessed. We detected both ARGs, mobile genetic elements and low rates of AMR. In particular, we observed four potential instances of bacterial clonal sharing in two different animal sources. This demonstrates resistant E. coli dissemination potential within the dairy farm, between calves and scavenger animals (rodents and flies). AMR dissemination and the zoonotic AMR risk is generally low in countries with low and restricted AMU. However, we show that interspecies dissemination does occur, and in countries that have little to no AMU restrictions this risk could be under-estimated.

Molecular Detection of Colistin Resistance mcr-1 Gene in Multidrug-Resistant Escherichia coli Isolated from Chicken.

Zoonotic and antimicrobial-resistant Escherichia coli (hereafter, E. coli) is a global public health threat which can lead to detrimental effects on human health. Here, we aim to investigate the antimicrobial resistance and the presence of mcr-1 gene in E. coli isolated from chicken feces. Ninety-four E. coli isolates were obtained from samples collected from different locations in Bangladesh, and the isolates were identified using conventional microbiological tests. Phenotypic disk diffusion tests using 20 antimicrobial agents were performed according to CLSI-EUCAST guidelines, and minimum inhibitory concentrations (MICs) were determined for a subset of samples. E. coli isolates showed high resistance to colistin (88.30%), ciprofloxacin (77.66%), trimethoprim/sulfamethoxazole (76.60%), tigecycline (75.53%), and enrofloxacin (71.28%). Additionally, the pathotype eaeA gene was confirmed in ten randomly selected E. coli isolates using primer-specific polymerase chain reaction (PCR). The presence of mcr-1 gene was confirmed using PCR and sequencing analysis in six out of ten E. coli isolates. Furthermore, sequencing and phylogenetic analyses revealed a similarity between the catalytic domain of Neisseria meningitidis lipooligosaccharide phosphoethanolamine transferase A (LptA) and MCR proteins, indicating that the six tested isolates were colistin resistant. Finally, the findings of the present study showed that E. coli isolated from chicken harbored mcr-1 gene, and multidrug and colistin resistance. These findings accentuate the need to implement strict measures to limit the imprudent use of antibiotics, particularly colistin, in agriculture and poultry farms.

A high-quality genome and comparison of short- versus long-read transcriptome of the palaearctic duck Aythya fuligula (tufted duck).

BACKGROUND: The tufted duck is a non-model organism that experiences high mortality in highly pathogenic avian influenza outbreaks. It belongs to the same bird family (Anatidae) as the mallard, one of the best-studied natural hosts of low-pathogenic avian influenza viruses. Studies in non-model bird species are crucial to disentangle the role of the host response in avian influenza virus infection in the natural reservoir. Such endeavour requires a high-quality genome assembly and transcriptome. FINDINGS: This study presents the first high-quality, chromosome-level reference genome assembly of the tufted duck using the Vertebrate Genomes Project pipeline. We sequenced RNA (complementary DNA) from brain, ileum, lung, ovary, spleen, and testis using Illumina short-read and Pacific Biosciences long-read sequencing platforms, which were used for annotation. We found 34 autosomes plus Z and W sex chromosomes in the curated genome assembly, with 99.6% of the sequence assigned to chromosomes. Functional annotation revealed 14,099 protein-coding genes that generate 111,934 transcripts, which implies a mean of 7.9 isoforms per gene. We also identified 246 small RNA families. CONCLUSIONS: This annotated genome contributes to continuing research into the host response in avian influenza virus infections in a natural reservoir. Our findings from a comparison between short-read and long-read reference transcriptomics contribute to a deeper understanding of these competing options. In this study, both technologies complemented each other. We expect this annotation to be a foundation for further comparative and evolutionary genomic studies, including many waterfowl relatives with differing susceptibilities to avian influenza viruses.

On-Site Pilot Testing of Hospital Wastewater Ozonation to Reduce Pharmaceutical Residues and Antibiotic-Resistant Bacteria.

Hospital sewage constitutes an important point source for antibiotics and antibiotic-resistant bacteria due to the high antibiotic use. Antibiotic resistance can develop and cause problems in sewage systems within hospitals and municipal wastewater treatment plants, thus, interventions to treat hospital sewage on-site are important. Ozonation has proven effective in treating relatively clean wastewater, but the effect on untreated wastewater is unclear. Therefore, we piloted implementation of ozonation to treat wastewater in a tertiary hospital in Uppsala, Sweden. We measured active pharmaceutical ingredients (APIs) using liquid chromatography-mass spectrometry and antibiotic-resistant Enterobacteriaceae using selective culturing pre- and post-ozonation. Comparing low (1 m3/h) and high (2 m3/h) flow, we obtained a 'dose-dependent' effect of API reduction (significant reduction of 12/29 APIs using low and 2/29 APIs using high flow, and a mean reduction of antibiotics of 41% using low vs. 6% using high flow, 25% vs. 6% for all APIs). There was no significant difference in the amount of antibiotic-resistant Enterobacteiaceae pre- and post-ozonation. Our results demonstrate that ozonation of untreated wastewater can reduce API content. However, due to the moderate API decrease and numerous practical challenges in the on-site setting, this specific ozonation system is not suitable to implement at full scale in our hospital.