Identification and evolution of ICE-PmuST394: a novel integrative conjugative element in Pasteurella multocida ST394.

BACKGROUND: The emergence of macrolide and tetracycline resistance within Pasteurella multocida isolated from feedlot cattle and the dominance of ST394 in Australia was reported recently. OBJECTIVES: To establish the genetic context of the resistance genes in P. multocida 17BRD-035, the ST394 reference genome, and conduct a molecular risk assessment of their ability to disperse laterally. METHODS: A bioinformatic analysis of the P. multocida 17BRD-035 genome was conducted to determine if integrative conjugative elements (ICEs) carrying resistance genes, which hamper antibiotic treatment options locally, are in circulation in Australian feedlots. RESULTS: A novel element, ICE-PmuST394, was characterized in P. multocida 17BRD-035. It was also identified in three other isolates (two ST394s and a ST125) in Australia and is likely present in a genome representing P. multocida ST79 from the USA. ICE-PmuST394 houses a resistance module carrying two variants of the blaROB gene, blaROB-1 and blaROB-13, and the macrolide esterase gene, estT. The resistance gene combination on ICE-PmuST394 confers resistance to ampicillin and tilmicosin, but not to tulathromycin and tildipirosin. Our analysis suggests that ICE-PmuST394 is circulating both by clonal expansion and horizontal transfer but is currently restricted to a single feedlot in Australia. CONCLUSIONS: ICE-PmuST394 carries a limited number of unusual antimicrobial resistance genes but has hotspots that facilitate genomic recombination. The element is therefore amenable to hosting more resistance genes, and therefore its presence (or dispersal) should be regularly monitored. The element has a unique molecular marker, which could be exploited for genomic surveillance purposes locally and globally.

Genomic profiling of Pasteurella multocida isolated from feedlot cases of bovine respiratory disease.

Pasteurella multocida causes a range of diseases in many host species throughout the world, including bovine respiratory disease (BRD) which is predominantly seen in feedlot cattle. This study assessed genetic diversity among 139 P. multocida isolates obtained from post-mortem lung swabs of BRD-affected feedlot cattle in four Australian states: New South Wales, Queensland, South Australia, and Victoria during 2014-2019. Whole-genome sequencing (WGS) was used to determine capsular serogroup, lipopolysaccharide genotypes, multi-locus sequence types and phylogenetic relationships. Two capsular types (A and D), with most isolates (132/139; 95%) belonging to type A; and three lipopolysaccharide (LPS) genotypes were identified (L1 [6/139; 4.3%], L3 [124/139; 89.2%] and L6 [9/139; 6.4%)]). Multi-locus sequence types (STs) ST9, ST13, ST17, ST20, ST36, ST50, ST58, ST79, ST124, ST125, ST132, ST167, ST185, ST327, ST394, and three novel STs [ST396, ST397, and ST398] were identified, with ST394 (59/139; 42.4%) and ST79 (44/139; 32%) the most prevalent in all four states. Isolates displaying phenotypic resistance to single, dual or multiple antibiotics (macrolide, tetracycline and aminopenicillins) were predominantly ST394 (23/139; 17%). Laterally mobile elements identified in the resistant ST394 isolates included small plasmids, encoding macrolide and/or tetracycline resistance, distributed in all states; and chromosomally located integrative conjugative elements (ICEs) (4 ST394 and 1 ST125) from the same Queensland feedlot. This study highlights the genomic diversity, epidemiological relationships and AMR associations in bovine P. multocida isolates from Australia and provides insight into the unique ST prevalence compared to other major beef-producing countries.

Antimicrobial susceptibility and molecular characteristics of Mycoplasma bovis isolated from cases of bovine respiratory disease in Australian feedlot cattle.

To date, antimicrobial susceptibility has not been reported for Australian Mycoplasma bovis isolates. This study determined minimal inhibitory concentrations (MICs) for 12 different antimicrobials against Australian M. bovis isolates and used whole genome sequencing to screen those showing high macrolide MICs for point mutations in target genes. Most lung tissue/swab samples from bovine respiratory disease cases (61/76, 80.3%) tested positive for M. bovis. A set of 50 representative isolates (50/61, 82.0%) that showed adequate growth, was used for MIC testing. Uniformly, low MIC values were confirmed for enrofloxacin (≤ 4 µg/mL), florfenicol (≤ 8 µg/mL), gamithromycin (≤ 2 µg/mL), spectinomycin (≤ 4 µg/mL), tetracycline (≤ 8 µg/mL), tiamulin (≤ 4 µg/mL), and tulathromycin (≤ 0.5 µg/mL). A small proportion (10%) of isolates exhibited high MICs (≥ 32 µg/mL) for tildipirosin, tilmicosin, tylosin, and lincomycin, which were above the epidemiological cut-off values for each antimicrobial (≥ 4 µg/mL). These isolates, originating from three Australian states, underwent whole genome sequencing/multilocus sequencing typing and were compared with the reference strain PG45 to investigate mutations that might be linked with the high macrolide/lincosamide MICs. All five belonged to ST52 and two macrolide associated mutations were identified within the 23 S rRNA gene (A2058G in two sequenced isolates and G748A in all sequenced isolates). Four additional 23 S rRNA gene mutations did not appear to be linked to macrolide resistance. Whilst the majority of Australian M. bovis isolates appear susceptible to the tested antimicrobials, emerging macrolide resistance was detected in three Australian states and requires continued monitoring.

Antimicrobial susceptibility and genomic analysis of Histophilus somni isolated from cases of bovine respiratory disease in Autralian feedlot cattle.

Histophilus somni is a prevalent commensal organism of the upper respiratory tract of cattle and a major causative agent of bovine respiratory disease (BRD) and other syndromes including myocarditis and infectious thromboembolic meningoencephalitis. This study investigated the antimicrobial susceptibility and phylogenetic relationships of H. somni isolates obtained from lung, heart, and other tissues at post-mortem as well as nasal mucosa swabs from cases of BRD in Australian feedlots (2004-2019). Broth microdilution Minimal Inhibitory Concentration (MIC) assays were determined for 19 antimicrobials using three different media (CLSI approved Veterinary Fastidious Medium [VFM], Mueller-Hinton fastidious broth medium supplemented with yeast extract [MHF-Y] and Columbia Broth [CB] supplemented with 5% lysed horse blood). For all antimicrobials, MICs obtained using CB medium were identical or within 1 dilution step of the MICs obtained for VFM and MHF-Y media. Therefore, CB may be a suitable medium for H. somni antimicrobial susceptibility testing similar to MHF-Y medium. None of the 70 Australian H. somni isolates exhibited resistance to antimicrobials with CLSI breakpoints including those commonly used in the treatment of BRD in Australia (first-line tetracyclines [chlortetracycline and oxytetracycline], second-line macrolides [tulathromycin], and third-line extended-spectrum cephalosporin [ceftiofur]). Whole-genome sequence analysis of 65 H. somni isolates for genomic single nucleotide polymorphism differences identified four phylogenetic clusters, each containing isolates from different Australian states, feedlots and tissue sources that clustered together. These findings demonstrate limited genetic diversity and the absence of significant antimicrobial resistance among Australian isolates of H. somni isolated from feedlot cattle.

Correction: Alhamami et al. First Emergence of Resistance to Macrolides and Tetracycline Identified in Mannheimia haemolytica and Pasteurella multocida Isolates from Beef Feedlots in Australia. Microorganisms 2021, 9, 1322.

The authors wish to make the following corrections to this paper [...].

Complete Genome Sequence of Pasteurella multocida Sequence Type 394, Isolated from a Case of Bovine Respiratory Disease in Australia.

Here, we present the completely closed genome sequence of Pasteurella multocida 17BRD-035, a bovine respiratory disease (BRD) pathogen from Queensland, Australia, with genes that confer resistance to ß-lactams, tilmicosin, and tetracycline. It consists of a single 2,624,884-bp chromosome and an average GC content of 40.23% and belongs to the newly described Rural Industries Research and Development Corporation (RIRDC) sequence type 394.

First Emergence of Resistance to Macrolides and Tetracycline Identified in Mannheimia haemolytica and Pasteurella multocida Isolates from Beef Feedlots in Australia.

Bovine respiratory disease (BRD) causes high morbidity and mortality in beef cattle worldwide. Antimicrobial resistance (AMR) monitoring of BRD pathogens is critical to promote appropriate antimicrobial stewardship in veterinary medicine for optimal treatment and control. Here, the susceptibility of Mannheimia haemolytica and Pasteurella multicoda isolates obtained from BRD clinical cases (deep lung swabs at post-mortem) among feedlots in four Australian states (2014-2019) was determined for 19 antimicrobial agents. The M. haemolytica isolates were pan-susceptible to all tested agents apart from a single macrolide-resistant isolate (1/88; 1.1%) from New South Wales (NSW). Much higher frequencies of P. multocida isolates were resistant to tetracycline (18/140; 12.9%), tilmicosin (19/140; 13.6%), tulathromycin/gamithromycin (17/140; 12.1%), and ampicillin/penicillin (6/140; 4.6%). Five P. multocida isolates (3.6%), all obtained from NSW in 2019, exhibited dual resistance to macrolides and tetracycline, and a further two Queensland isolates from 2019 (1.4%) exhibited a multidrug-resistant phenotype to ampicillin/penicillin, tetracycline, and tilmicosin. Random-amplified polymorphic DNA (RAPD) typing identified a high degree of genetic homogeneity among the M. haemolytica isolates, whereas P. multocida isolates were more heterogeneous. Illumina whole genome sequencing identified the genes msr(E) and mph(E)encoding macrolide resistance, tet(R)-tet(H) or tet(Y) encoding tetracycline resistance, and blaROB-1 encoding ampicillin/penicillin resistance in all isolates exhibiting a corresponding resistant phenotype. The exception was the tilmicosin-resistant, tulathromycin/gamithromycin-susceptible phenotype identified in two Queensland isolates, the genetic basis of which could not be determined. These results confirm the first emergence of AMR in M. haemolytica and P. multocida from BRD cases in Australia, which should be closely monitored.

Globoid cell leukodystrophy (Krabbe disease) in a Merino sheep.

We describe the clinicopathologic features of an ovine case of Krabbe disease (globoid cell leukodystrophy). Brain lesions, sometimes bilaterally distributed, were present in the cerebellar peduncles, cerebellar folia white matter, medulla, pons, and spinal cord and characterized by marked myelin loss and numerous large macrophages (globoid cells), which tended to aggregate perivascularly. Gemistocytic astrocytes were abundant, and their nuclei were frequently abnormal. The activity of the deficient enzyme, galactosylceramide ß-galactosidase, was undetectable in this neurologic disorder compared to age- and breed-matched control brains, and levels of the neurotoxic substrate, psychosine, were markedly elevated.