A 2-day-old male Belgian blue calf (45 kg) was presented in December 2018 in a critically ill state. The calf had experienced breathing difficulties since the first day after birth. The calf had been treated by the local practitioner with furosemide, amoxicillin, dexamethasone, alpha tocopherol acetate and anhydrous sodium selenite, presumably all intramuscularly as indicated by the manufacturer, except for the furosemide, which was likely given intravenously. At presentation, the calf was in lateral decubitus and depressed. Fever (40.8 C: ref. 38.5-39.5) was present, with an increased respiratory rate (72/min: ref. 20-50), but heart rate (108/min: ref. 90-110) was normal. Ultrasonography was performed with a linear 7.5 MHz probe (Easote MyLab 30 Gold unit, the Netherlands) and revealed diffuse comet tail artifacts (B-lines) on the pleura and multiple small consolidations (max. 1 cm depth) on both sides of the thorax. No abnormalities were found on ultrasonographic examination of the abdomen. Blood samples were collected from the jugular vein upon arrival and placed in heparin-coated tubes prior to blood-gas analysis with RAPIDPoint  405 (Siemens Healthcare, Beersel, Belgium). Venous blood gas analysis showed a mild metabolic acidosis (pH 7.23). The morning after presentation, an arterial sample out of the external carotid artery displayed hypoxemia [arterial pO2 = 32.6 mmHg (ref. 80-100)] and hypercapnia [arterial pCO2 = 74.7 mmHg (ref. 40-50)]. Failure of passive transfer of immunoglobulins was excluded with a glutaraldehyde test according to Turgut et al..
Blood samples were aseptically sampled in one single volume collected from the jugular vein upon arrival, whereby 3 and 10 ml were aseptically inoculated into BD BACTEC  Peds Plus  and BD BACTEC  Plus Aerobic medium, respectively (BD, Erembodegem, Belgium), which were subsequently incubated at 35 C in an automated system for the detection of microbial growth (BACTEC  FX). For identification, a loopful of medium was first plated on a Columbia agar supplemented with 5% sheep blood (blood agar; Oxoid, Hampshire, UK) and incubated for 24 h at 37 C in a 5% CO2 enriched atmosphere, after which a single colony was transferred on a polished steel plate, air dried and covered with 1 muL alpha-cyano-4-hydroxycinnamic acid matrix (Bruker Daltonics, Bremen, Germany). The sample was identified with an Autoflex III smartbeam MALDI-TOF MS, using FlexControl and MBT Compass software (Bruker Daltonics, Bremen, Germany). Log score values higher than 2.00, between 1.70 and 1.99, and between 0 and 1.69, indicate high-confidence, low-confidence, and no-confidence identification, respectively. From both BD BACTEC  media (Becton Dickinson:Benelux, Erembodegem, Belgium), a pure culture was obtained. The isolate was identified with MALDI-TOF MS, and the best hit was obtained for R. ornithinolytica MB_18887 CHB with a (log)score value of 2.32. The seven best hits matched with R. ornithinolytica, with (log)score values >=2.16.
Antimicrobial susceptibility testing (AST) for different antibiotics (Table 1) was executed with the disk diffusion test using a Mueller Hinton agar and aerobic incubation at 35 C for 20 h after which the inhibition zones were interpreted. The diameter of the inhibition zone was measured and interpreted according to the breakpoints prescribed by the Clinical and Laboratory Standards Institute (CLSI). CLSI breakpoints specifically for R. ornithinolytica were not available, so CLSI values were used for Enterobacterales, since R. ornithinolytica belongs to the Enterobacteriaceae family. For ceftiofur, cattle-specific disk diffusion CLSI breakpoints were available. For the other antibiotics, CLSI breakpoints extrapolated from human breakpoints were used, rather than extrapolation from other organisms. In the absence of CLSI values for Enterobacterales for certain antibiotics (or certain potencies), breakpoints provided by the manufacturer were used (Table A1 in Appendix). Escherichia coli strains ATCC  25922  and ATCC  35218  were used as quality control strains. Multidrug resistance was defined as resistance to agents from at least three different antimicrobial classes. The isolate showed multidrug resistance with the disk diffusion test, with resistance against ampicillin, tetracycline and doxycycline, gentamicin, potentiated sulfonamides and streptomycin and intermediate susceptibility for enrofloxacin (Table 1 and Table A1 in Appendix).
The patient was suspected to have neonatal respiratory distress syndrome (NRDS) combined with sepsis. Based on the clinical presentation of the animal, therapy initialization could not be post-poned until microbial information was present. The calf was empirically treated with enrofloxacin (Floxadil  5 mg/kg, Emdoka, Hoogstraten, Belgium), sodium benzylpenicillin (penicillin 1.500.000 IU q.i.d., Kela, Sint-Niklaas, Belgium) and oxygen (1.5 L/min.) for seven days, ketoprofen (Ketofen  3 mg/kg, Ceva, Libourne, France) during the first 2 days and doxapram hydrochloride (Dopram  1.5 mg/kg, Eumedica, Brussel, Belgium) on day one. Perfusions were given intravenously through a catheter, starting with bicarbonate 2% (20 gram bicarbonate in 1 liter of isotonic NaCl 0.9%; rate 100 mL/kg/h), followed by Ringer's lactate solution at a rate of 20 mL/kg/h. The calf received perfusions until it drank sufficiently on day 6. After ultrasonography and clinically improvement, the calf could leave the clinic.
For research purposes, the isolated was further characterized by whole genome sequencing (WGS). Genomic DNA was prepared using the Isolate II Genomic DNA kit (Bioline, Meridian Bioscience, Paris, France), following the manufacturer's instructions. Sequencing libraries were constructed using the Illumina Nextera XT DNA sample preparation kit and subsequently sequenced on an Illumina MiSeq instrument with a 250-bp paired end protocol (MiSeq v3 chemistry) according to the manufacturer's instructions. Generated WGS data have been deposited in the NCBI Sequence Read Archive (SRA) under accession number PRJNA607902. For species identification based on WGS data, three different approaches were used: k-mer based identification of read data using Kraken against the entire NCBI RefSeq Microbial Genomes database, 16S rRNA gene analysis using the NCBI suite of tools, and read mapping using Bowtie2 against the NCBI RefSeq reference genome entry for R. ornithinolytica (NCBI accession NC_021066.1). For genotypic antimicrobial resistance (AMR) gene detection, WGS data were searched using an in-house workflow based on assembly with SPAdes and BLASTing against the ResFinder database containing a curated catalog of AMR genes.
Mlplasmids 1.0.0 (https://sarredondo.shinyapps.io/mlplasmids) was used to determine whether the predicted contig origin was plasmid- or chromosome-derived. Klebsiella pneumoniae was used as species model. Additionally, a direct read mapping approach was performed using SRST2 against the ResFinder database. One of the detected isolate AMR genes, blaPLA, is a genetic marker that allows differentiation of Raoultella spp., and was further investigated using the NCBI suite of tools. A more extended description of bioinformatics methods for WGS data analysis including tools, versions, and parameters, is available in the Supplementary Material.
Identification of the isolate as R. ornithinolytica was supported by WGS data through three different analyses. Firstly, k-mer based identification of WGS data against the NCBI RefSeq Microbial Genomes database classified 87.52% of reads as R. ornithinolytica, whereas no other species were detected using a 5% threshold (see Supplementary Figure 1). Secondly, 16S rRNA analysis using the NCBI RefSeq 16S database confirmed the presence of a 16S rRNA R. ornithinolytica gene with high sequence identity (>99%). Lastly, read mapping against the NCBI RefSeq Genome entry for R. ornithinolytica confirmed its presence with a depth and breadth of coverage of 30.52x and 94.52%, respectively (see Supplementary Figure 2). Genotypic AMR gene detection using the ResFinder database, both through an assembly- and read mapping-based approach, indicated the presence of several AMR genes consistently with both methods and various of the detected genes were predicted to be present on a plasmid (see Table 1). Isolate AMR genes that contained mutations compared to the ResFinder reference sequences were manually investigated. The isolate's fosA gene contained two synonymous mutations, and the isolate's aph(3")-Ib contained one mutation resulting in a K10E amino acid change, preserving the open reading frame of both isolate genes. The isolate's blaPLA gene, encoding a class A broad-spectrum beta-lactamase originally characterized from R. planticola, exhibited however 49 mutations. Because this gene acts as a species marker for Raoultella spp., the sequence of the isolate's AMR gene was further investigated, finding only one synonymous mutation compared to the blaORN gene from R. ornithinolytica, confirming the functional presence of blaORN and isolate identification as R. ornithinolytica (see Supplementary Figures 3, 4). A more extended description of results for the WGS data analysis is available in the Supplementary Material.