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 β-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

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 [1]: There are metadata errors in Supplementary Figure S2 and Table 8, which state that isolate 17BRD-035 was isolated from a feedlot in NSW when in fact it came from Queensland. Figure S2 in Supplementary Materials was changed accordingly and was included with a separate document: https://www.mdpi.com/article/10.3390/microorganisms9061322/s1 In Table 8, the ST column for strain P. m 17BRD-035 was changed from NSW to QLD, the correct Table 8 is as follows: Table 8. Resistance profile, RAPD pattern and presence of antimicrobial resistance genes among isolates of Pasteurella multocida (P. m) (n = 28) and Mannheimia haemolytica (M. h) (n = 1) +, present, −, absent. Table (see supplement) The authors apologize for any inconvenience caused and state that the scientific conclusions are unaffected. The original article has been updated

Phenotypic and Genetic Analysis of Antimicrobial Resistance in Bovine Respiratory Disease Pathogens from Australian Feedlots

The use of antimicrobials in Australian beef feedlots is essential for preventing disease, as well as for modifying the rumen for grain-based diets to reduce greenhouse gases. The purpose of regular monitoring of antimicrobial resistance through surveillance is to maintain the lifespan of currently registered antimicrobial classes, and in particular, shared drug classes (i.e. those registered for both human and animal treatment). In contrast, new antimicrobial drug classes are only being developed for the human market. It is critical that the Australian feedlot industry understands and embraces antimicrobial stewardship guidelines to continue to effectively treat and prevent bacterial infections in cattle. Bovine Respiratory Disease (BRD) is the major cause of morbidity and mortality in feedlot cattle, both in Australia and internationally. BRD has been described as a composite of infections involving the respiratory system of cattle. The main bacterial causes of BRD are Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni, and antimicrobial agents used to treat BRD in Australia include tulathromycin, tetracycline, tilmicosin, and ceftiofur. Internationally, antimicrobial resistance (AMR) has rapidly developed in BRD pathogens in North America due to the widespread dissemination of mobile genetic elements referred to as Integrative Conjugative Elements (ICEs) containing multiple resistance genes. Unfortunately, there is very little research on antimicrobial resistance in BRD pathogens in Australian feedlots, and only two previous studies (1993 and 2014). The main aim of this thesis was to determine the antimicrobial susceptibility of Australian BRD isolates obtained between 2004-2019, determine the genetic basis of resistance, and undertake a molecular epidemiological investigation. A preliminary investigation on the susceptibility of a small collection of BRD pathogens, obtained from diagnostic submissions to veterinary diagnostic laboratories (VDLs) between 2004 and 2016, to gamithromycin, a new generation veterinary macrolide was firstly undertaken. No gamithromycin resistance was identified and antimicrobial susceptibility testing (AST) methodology was improved for this antimicrobial. Further BRD isolates were obtained from VDLs between 2016-2018 and a prospective study was conducted at eight Australian feedlots during 2019. AST was undertaken through MIC determination and whole-genome sequencing (WGS) was employed to identify antimicrobial resistance genes (ARGs) and determine phylogenetic relationships. Minimum inhibitory concentration (MIC) values were determined for M. haemolytica (n= 88), P. multocida (n=140) and H. somni (n=70) obtained from post-mortem samples at feedlots in New South Wales (NSW), Queensland (QLD), Tasmania (TAS), Victoria (VIC), and South Australia (SA). All M. haemolytica were susceptible to the 19 tested antimicrobials except for a single 2019 isolate from NSW that was resistant to macrolides (1/88; 1.1%). H. somni isolates were susceptible to all tested antimicrobials. Resistance was most prevalent in P. multocida to tetracycline (18/140; 12.9%), tilmicosin (19/140;13.6%), tulathromycin/gamithromycin (17/140; 12.1%), and ampicillin/penicillin (6/140; 4.6%). A multidrug-resistant phenotype (aminopenicillin-tetracycline-tilmicosin resistance) was identified in five Queensland isolates from a single feedlot, providing an early indication of the presence of possible mobile genetic elements. Among the isolates, genes imparting resistance to macrolides (mrs(E) and mph(E)), tetracycline (tet(R)-tet(H) or tet(Y)) and ampicillin/penicillin (blaROB-1) were found, with ARGs identified in all isolates exhibiting a resistance phenotype. Increases in the frequency of tetracycline and macrolide resistance were found in 2019 isolates compared with 2016-2018 isolates. While these resistance levels are low by international standards, the results confirm Australia’s first case of AMR to frontline antimicrobials used to treat BRD. Molecular epidemiology of the M. haemolytica and P. multocida isolates was initially investigated using random-amplified polymorphic DNA (RAPD) analysis followed by WGS and phylogenetic comparisons for P. multocida and H. somni. Whilst a high degree of genetic homogeneity was identified among the isolates of M. haemolytica and H. somni, significant phylogenetic diversity was identified among the P. multocida isolates. Two capsular types (A [95%] and D [5%]), and three lipopolysaccharide (LPS) genotypes (L1 (6/139; 4.3%), L3 (124/139; 89.2%) and L6 (9/139; 6.4%) were identified. Newly identified multilocus sequence type (ST)394 (60/139; 43.1%) was the most prevalent ST within the A: L3 clonal lineage and contained a higher prevalence of resistant isolates, including those exhibiting dual resistance to macrolides and tetracyclines and the tilmicosin-tetracycline-ampicillin/penicillin phenotype. The other major ST identified by WGS was ST79 (44/139; 31.7%) and less predominant STs included ST9, ST13, ST17, ST20, ST50, ST58, ST79, ST124, ST125, ST132, ST167, ST185, ST327, and 3 novel STs (ST396, ST397 and ST398). Deeper analysis of genomic data suggested the presence of distinct plasmids encoding macrolides and tetracycline and a putative ICE, with long range sequencing now required. This study confirms the emergence of significant AMR in P. multocida and M. haemolytica isolated from BRD cases in Australia. This suggests that a rotation of antimicrobials (including florfenicol) should be considered, along with non-antimicrobial management practices to limit AMR spread. Development of an efficient vaccination against P. multocida would be a vital tool in decreasing the substantial economic losses associated with this major BRD pathogen affecting Australian feedlots. Continued AMR monitoring is needed to support antimicrobial stewardship programs recently developed by the industry that are designed to ensure the continued effectiveness of first and second-line veterinary drugs.Thesis (Ph.D.) -- University of Adelaide, School of Animal and Veterinary Sciences, 202

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

The emergence of macrolide and tetracycline resistance within Pasteurella multocida isolated from feedlot cattle and the dominance of ST394 in Australia was reported recently.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.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.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.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