Examining antibiotic resistance in the feedlot cattle industry using real-time, quantitative PCR (qPCR) and enterococci as an indicator bacterium

Antibiotics are administered to livestock at subtherapeutic levels to maintain animal health. Many of the antibiotics used are analogues or the same as those used in human medicine, raising the possibility that genes conferring resistance arise within agricultural production systems. This thesis examined antibiotic resistance in the Canadian beef feedlot industry. Real-time, quantitative PCR was used to examine differences in the relative abundance of eighteen resistance genes across five antibiotic families from feedlot cattle faeces and urban environments. The effect of infeed administration and withdrawal of tylosin phosphate on macrolide resistance was examined using enterococci as an indicator bacterium. Resistant enterococci (n=21) were selected for whole-genome sequencing and comparative genomics. Results presented here show that the relative abundance of resistance genes differs between cattle feedlots and urban environments, likely a reflection of differences in antibiotic use. Sulfonamide, fluoroquinolone and β-lactam resistance genes predominated in urban wastewater, whilst tetracycline resistance genes were more prevalent in cattle faeces. Macrolide use in cattle production increased the proportion of erythromycinand tylosin-resistant enterococci. However, withdrawal of tylosin from the diet appeared to contribute to reduced macrolide resistance in enterococci. Comparative genomics revealed resistance to macrolides was present on mobile genetic elements, specifically the Tn917 transposon harbouring erm(B). This transposon was identified in both Enterococcus hirae and Enterococcus faecium suggesting inter-species transfer of resistance genes may occur in the bovine gastrointestinal tract. Furthermore, the integrative conjugative elements (ICEs) Tn916 and Tn5801, both conferring tetracycline resistance, were identified in E. faecium. As the cost of genomic sequencing continues to decrease, further investigation of ICEs using whole genome sequencing will help determine if there are linkages between enterococci isolates from bovine environmental and human clinical sources and whether bovine enterococci represent a source to the dissemination and spread of antibiotic resistance

Peri-operative red blood cell transfusion in neonates and infants: NEonate and Children audiT of Anaesthesia pRactice IN Europe: A prospective European multicentre observational study

BACKGROUND: Little is known about current clinical practice concerning peri-operative red blood cell transfusion in neonates and small infants. Guidelines suggest transfusions based on haemoglobin thresholds ranging from 8.5 to 12 g dl-1, distinguishing between children from birth to day 7 (week 1), from day 8 to day 14 (week 2) or from day 15 (≥week 3) onwards. OBJECTIVE: To observe peri-operative red blood cell transfusion practice according to guidelines in relation to patient outcome. DESIGN: A multicentre observational study. SETTING: The NEonate-Children sTudy of Anaesthesia pRactice IN Europe (NECTARINE) trial recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. PATIENTS: The data included 5609 patients undergoing 6542 procedures. Inclusion criteria was a peri-operative red blood cell transfusion. MAIN OUTCOME MEASURES: The primary endpoint was the haemoglobin level triggering a transfusion for neonates in week 1, week 2 and week 3. Secondary endpoints were transfusion volumes, 'delta haemoglobin' (preprocedure - transfusion-triggering) and 30-day and 90-day morbidity and mortality. RESULTS: Peri-operative red blood cell transfusions were recorded during 447 procedures (6.9%). The median haemoglobin levels triggering a transfusion were 9.6 [IQR 8.7 to 10.9] g dl-1 for neonates in week 1, 9.6 [7.7 to 10.4] g dl-1 in week 2 and 8.0 [7.3 to 9.0] g dl-1 in week 3. The median transfusion volume was 17.1 [11.1 to 26.4] ml kg-1 with a median delta haemoglobin of 1.8 [0.0 to 3.6] g dl-1. Thirty-day morbidity was 47.8% with an overall mortality of 11.3%. CONCLUSIONS: Results indicate lower transfusion-triggering haemoglobin thresholds in clinical practice than suggested by current guidelines. The high morbidity and mortality of this NECTARINE sub-cohort calls for investigative action and evidence-based guidelines addressing peri-operative red blood cell transfusions strategies. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT02350348

Effect of in-feed administration of tylosin phosphate on antibiotic resistance in enterococci isolated from feedlot steers

Tylosin phosphate is a macrolide commonly administered to cattle in North America for the control of liver abscesses. This study investigated the effect of in-feed administration of tylosin phosphate to cattle at subtherapeutic levels and its subsequent withdrawal on macrolide resistance using enterococci as an indicator bacterium. Faecal samples were collected from steers that received no antibiotics and steers administered tylosin phosphate (11 ppm) in-feed for 197 d and withdrawn 28 d before slaughter. Enterococcus species isolated from faecal samples were identified through sequencing the groES-EL intergenic spacer region and subject to antimicrobial susceptibility testing, identification of resistance determinants and pulsed-field gel electrophoresis profiling. Tylosin increased (P 0.05) between treatments on d 225. This suggests that antibiotic withdrawal prior to slaughter contributes to a reduction in the proportion of macrolide resistant enterococci entering the food chain. Among the 504 enterococci isolates characterised, E. hirae was found to predominate (n=431), followed by E. villorum (n=32), E. faecium (n=21), E. durans (n=7), E. casseliflavus (n=4), E. mundtii (n=4), E. gallinarum (n=3), E. faecalis (n=1), and E. thailandicus (n=1). The diversity of enterococci was greater in steers at arrival than at exit from the feedlot. Erythromycin resistant isolates harboured the erm(B) and/or msrC gene. Similar PFGE profiles of eryR E. hirae n pre- and post-antibiotic treatment suggests the increased abundance of eryR enterococci after administration of tylosin phosphate reflects selection for strains that were within the bovine gastrointestinal tract of cattle at arrival

Cronobacter sakazakii Infection from Expressed Breast Milk, Australia

Cronobacter sakazakii neonatal infections are often epidemiologically linked to the consumption of contaminated powdered infant formula. We describe a case resulting from consumption of contaminated expressed breast milk, as confirmed by whole-genome sequencing. This case highlights potential risks associated with storage and acquisition of expressed breast milk

Failure of daptomycin β-Lactam combination therapy to prevent resistance emergence in Enterococcus faecium

Daptomycin β-Lactam combination therapy offers "protection" against daptomycin non-susceptibility (DNS) development in Enterococcus faecium. We report failure of this strategy and the importance of source control. Mutations were detected in the LiaF and cls genes in DNS isolates. A single DNS isolate contained an unrecognized mutation, which requires confirmation

Additional file 2: Figure S1. of Comparative genomics of Enterococcus spp. isolated from bovine feces

Organisation of protein coding genes by Clusters of Orthologous Groups (COGs) category. (PNG 731 kb

Analytical validity of nanopore sequencing for rapid SARS-CoV-2 genome analysis

ABSTRACT Viral whole-genome sequencing (WGS) provides critical insight into the transmission and evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Long-read sequencing devices from Oxford Nanopore Technologies (ONT) promise significant improvements in turnaround time, portability and cost, compared to established short-read sequencing platforms for viral WGS (e.g., Illumina). However, adoption of ONT sequencing for SARS-CoV-2 surveillance has been limited due to common concerns around sequencing accuracy. To address this, we performed viral WGS with ONT and Illumina platforms on 157 matched SARS-CoV-2-positive patient specimens and synthetic RNA controls, enabling rigorous evaluation of analytical performance. Despite the elevated error rates observed in ONT sequencing reads, highly accurate consensus-level sequence determination was achieved, with single nucleotide variants (SNVs) detected at >99% sensitivity and >98% precision above a minimum ~60-fold coverage depth, thereby ensuring suitability for SARS-CoV-2 genome analysis. ONT sequencing also identified a surprising diversity of structural variation within SARS-CoV-2 specimens that were supported by evidence from short-read sequencing on matched samples. However, ONT sequencing failed to accurately detect short indels and variants at low read-count frequencies. This systematic evaluation of analytical performance for SARS-CoV-2 WGS will facilitate widespread adoption of ONT sequencing within local, national and international COVID-19 public health initiatives

Respiratory viral co-infections among SARS-CoV-2 cases confirmed by virome capture sequencing

Accumulating evidence supports the high prevalence of co-infections among Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) patients, and their potential to worsen the clinical outcome of COVID-19. However, there are few data on Southern Hemisphere populations, and most studies to date have investigated a narrow spectrum of viruses using targeted qRT-PCR. Here we assessed respiratory viral co-infections among SARS-CoV-2 patients in Australia, through respiratory virome characterization. Nasopharyngeal swabs of 92 SARS-CoV-2-positive cases were sequenced using pan-viral hybrid-capture and the Twist Respiratory Virus Panel. In total, 8% of cases were co-infected, with rhinovirus (6%) or influenzavirus (2%). Twist capture also achieved near-complete sequencing (> 90% coverage, > tenfold depth) of the SARS-CoV-2 genome in 95% of specimens with C