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Refereed/Peer-reviewed 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.

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.

Desymmetrisation of robenidine (1: N ',2-bis((E)-4-chlorobenzylidene)hydrazine-1-carboximidhydrazide) and the introduction of imine alkyl substituents gave good antibiotic activity. Of note was the increased potency of two analogues against vancomycin-resistant Enterococci (VRE), one of which returned a MIC of 0.5 mu g mL(-1). Five analogues were found to be equipotent or more potent than the lead 1. Introduction of an indole moiety resulted in the most active robenidine analogue against methicillin-resistant S. aureus (MRSA), with a MIC of 1.0 mu g mL(-1). Imine C=NH isosteres (C=O/C=S) were inactive. Monomeric analogues were 16-64 mu g mL(-1) active against MRSA and VRE. An analogue that lacks the terminal hydrazide NH moiety showed modest Gram-negative activity at 64 mu g mL(-1). A 4-tert-butyl analogue was shown to be active against both Gram-positive and -negative strains at 16-64 mu g mL(-1). In general, additional modifications with aromatic moieties was poorly tolerated, except with concomitant introduction of an imine C-alkyl group. The activity of these analogues against MRSA and VRE ranged from 8 mu g mL(-1) to inactive (MIC>128 mu g mL(-1)) with the naphthyl and indole analogues. Gram-negative activity was most promising with two compounds at 16 mu g mL(-1) against E. coli. Against P. aeruginosa, the highest activity observed was with MIC values of 32 mu g mL(-1) with another two analogues. Combined, these findings support the further development of the (E)-2-benzylidenehydrazine-1-carboximidamide scaffold as a promising scaffold for the development of antibiotics against Gram-positive and Gram-negative strains. Refereed/Peer-reviewed

Stone formation and catheter blockage are major complications of Proteus UTIs. In this study, we investigated the ability of allicin to inhibit P. mirabilis-induced struvite crystallization and catheter blockage using a synthetic bladder model. Struvite crystallization inhibition study was carried out using P. mirabilis lysate as urease enzyme source in synthetic urine (SU). Struvite productions were monitored by phase contrast light microscopy and measurements of pH, Mg 2+ and Ca 2+ precipitation and turbidity. A catheter blockage study was performed in a synthetic bladder model mimicking natural UTI in the presence of allicin at sub-MIC concentrations (MIC = 64 μg/ml). The results of crystallization study showed that allicin inhibited pH rise and consequently turbidity and precipitation of ions in a dose-dependent manner. The results of catheter blockage study showed that allicin at sub-MIC concentrations (2, 4, 8 μg/ml) significantly increased the time for catheter blockage to occur to 61, 74 and 92 h respectively compared to allicin-free control (48 h). In a similar way, the results showed that allicin delayed the increase of SU pH level in bladder model in a dose-dependent manner compared to allicin-free control. The results also showed that following the increase of allicin concentration, Mg 2+ and Ca 2+ deposition in catheters were much lower compared to allicin-free control, further confirmed by direct observation of the catheters’ eyehole and cross sections. We conclude that allicin prevents the formation of Proteus-induced urinary crystals and the blockage of catheters by delaying pH increase and lowering Mg 2+ and Ca 2+ deposition in a dose-dependent manner. Refereed/Peer-reviewed

Multidrug-resistant pathogens present a major global challenge in antimicrobial therapy and frequently complicate otitis externa in dogs.In vitro efficacy of oregano oil, thyme oil and their main phenolic constituents against bacterial and fungal isolates associated with canine otitis externa were investigated. It was hypothesized that the main phenolic components would have greater antimicrobial activity compared to the relative essential oil.Antimicrobial susceptibility testing was performed using broth microdilution with spot-plating technique to determine minimum inhibitory and bactericidal/fungicidal concentrations (MICs, MBCs and MFCs). A time-kill kinetics assay was performed to confirm the bactericidal and fungicidal activity of the oils and their phenolic constituents. One hundred bacterial and fungal isolates, including meticillin-susceptible Staphylococcus pseudintermedius (n = 10), meticillin-resistant S. pseudintermedius (n = 10), β-haemolytic Streptococcus spp. (n = 20), Pseudomonas aeruginosa (n = 20; including 10 isolates resistant to one or two antimicrobials), Proteus mirabilis (n = 20) and Malassezia pachydermatis (n = 20) from dogs with otitis externa were used.Oregano oil, thyme oil, carvacrol and thymol exhibited antibacterial activity against all bacterial and fungal isolates tested. MICOregano oil, thyme oil, carvacrol and thymol showed good in vitro bactericidal and fungicidal activity against 100 isolates from dogs with otitis externa, including some highly drug-resistant isolates. These essential oils and their main phenolic constituents have the potential to be further investigated in vivo for the treatment of canine otitis externa.Les pathogènes multirésistants représentent un défi majeur pour la thérapeutique antimicrobienne et compliquent fréquemment les otites externes canines. HYPOTHÈSES/OBJECTIFS: Nous avons étudiés l'efficacité in vitro de l'huile d'origan, de l'huile de thym et de leurs principaux composés phénoliques, contre les souches bactériennes et fongiques associées aux otites externes canines. Il est supposé que les composés phénoliques principaux auraient une meilleure activité antimicrobienne en comparaison avec les huiles essentielles. MATÉRIELS ET MÉTHODES: La sensibilité antimicrobienne a été testée par microdilution sur plaque pour déterminer la concentration minimale inhibitrice et les concentrations bactéricides/fongicides (MICs, MBCs et MFCs). Un test cinétique temps dépendant a été réalisé pour confirmer l'activité antifongique et bactéricide des huiles et de leurs constituants phénoliques. Cent souches bactériennes et fongiques ont été utilisées, comprenant Staphylococcus pseudintermedius sensible à la méticiline (n = 10), S. pseudintermedius résistante à la méticiline (n = 10), Streptococcus spp. β-hémolytique (n = 20), Pseudomonas aeruginosa (n = 20; comprenant 10 souches résistantes à un ou deux antimicrobiens), Proteus mirabilis (n = 20) et Malassezia pachydermatis (n = 20) de chiens avec otite externe. RÉSULTATS: L'huile d'origan, l'huile de thym, le carvacrol et le thymol ont montré une activité antibactérienne contre toutes les souches bactériennes et fongiques testées. Les valeurs de MIC90 allant de 0.015 à 0.03% (146-292 μg/mL) pour la bactérie Gram-positive et P. mirabilis. Pour P. aeruginosa et M. pachydermatis, les valeurs de MIC90 allaient de 0.09 à 0.25% (800-2,292 μg/mL).L'huile d'origan, de thym, le carvacrol et le thymol ont montré une bonne activité bactéricide et fongicide contre 100 souches de chiens avec otite externe, comprenant des souches hautement résistantes. Ces huiles essentielles et leurs constituants phénoliques principaux ont le potentiel d’être étudiés in vivo pour le traitement des otites externes canines.INTRODUCCIÓN: los patógenos resistentes a múltiples fármacos presentan un reto global importante en la terapia antimicrobiana y con frecuencia complican la otitis externa en perros. HIPÓTESIS/OBJETIVOS: Se investigó la eficacia in vitro del aceite de orégano, el aceite de tomillo y sus principales componentes fenólicos contra los aislados bacterianos y fúngicos asociados con la otitis externa canina. Se planteó la hipótesis de que los principales componentes fenólicos tendrían una mayor actividad antimicrobiana en comparación con el aceite esencial relativo. MÉTODOS Y MATERIALES: las pruebas de susceptibilidad a los antimicrobianos se realizaron mediante microdilución en caldo con técnica de cultivo puntual para determinar las concentraciones mínimas inhibitorias y bactericidas/fungicidas (MIC, MBC y MFC). Se realizó un ensayo de cinética de tiempo de destrucción para confirmar la actividad bactericida y fungicida de los aceites y sus componentes fenólicos. Cien aislados bacterianos y fúngicos, incluyendo Staphylococcus pseudintermedius susceptible a meticilina (n = 10), S. pseudintermedius resistente a meticilina (n = 10), Streptococcus sp. β-hemolítico (n = 20), Pseudomonas aeruginosa (n = 20; incluidos 10 aislamientos resistentes a uno o dos antimicrobianos), Proteus mirabilis (n = 20) y Malassezia pachydermatis (n = 20) de perros con otitis externa. RESULTADOS: el aceite de orégano, el aceite de tomillo, el carvacrol y el timol exhibieron actividad antibacteriana contra todos los aislamientos bacterianos y fúngicos probados. Los valores de MICMultiresistente Pathogene stellen eine große globale Herausforderung bei der antimikrobiellen Therapie dar und verkomplizieren häufig eine Otitis externa von Hunden.Die in vitro Wirksamkeit von Oreganoöl, Thymianöl und ihrer hauptsächlichen phenolischen Bestandteile gegen bakterielle und mykologische Isolate, die mit einer Otitis externa im Zusammenhang stehen, wurden untersucht. Es wurde die Hypothese aufgestellt, dass die hauptsächlichen Phenol-Komponenten eine größere antimikrobielle Aktivität im Vergleich zu den relativen essentiellen Ölen haben.Es wurden Hemmstoffnachweise mittels Mikrodilutionsbouillon mit einer Spot- Plating Technik durchgeführt, um eine minimale Hemmstoffkonzentration und bakterielle/fungizide Konzentrationen (MICs, MBCs und MFCs) zu bestimmen. Ein Time-Kill Kinetik Assay wurde durchgeführt, um bakterizide und fungizide Aktivitäten der Öle und ihrer phenolischen Bestandteile zu bestätigen. Es wurden einhundert bakterielle und mykologische Isolate, inklusive Methicillin-empfindlichem Staphylococcus pseudintermedius (n = 10), Methicillin-resistentem S. pseudintermedius (n = 10), β-hämolytischem Streptococcus spp. (n = 20), Pseudomonas aeruginosa (n = 20; inklusive 10 Isolaten, die resistent zu einem oder zwei antimikrobiellen Wirkstoffen waren), Proteus mirabilis (n = 20) und Malassezia pachydermatis (n = 20) von Hunden mit einer Otitis externa verwendet.Oreganoöl, Thymianöl, Carvacrol und Thymol zeigten eine antibakterielle Aktivität gegenüber allen getesteten bakteriellen und mykologischen Isolaten. MICOreganoöl, Thymianöl, Carvacrol und Thymol zeigten eine gute in vitro bakterizide und fungizide Aktivität gegenüber 100 Isolaten von Hunden mit Otitis externa, wobei einige hochresistente Isolate dabei waren. Diese essentiellen Öle und ihre hauptsächlichen phenolischen Bestandteile haben das Potential in vivo für die Therapie einer Otitis externa des Hundes weiter untersucht zu werden.背景: 多剤耐性病原体は、抗菌薬治療における世界的な主要課題であり、犬外耳炎をしばしば複雑化する。 仮説/目的: 本研究の目的は、犬の外耳炎に関連する細菌および真菌分離株に対するオレガノ油、タイム油、およびそれらの主要フェノール成分のin vitroにおける有効性を調査することであった。主要なフェノール成分は、関連精油と比較してより高い抗菌活性を持つと仮定した。 材料と方法: 最小発育阻止濃度および殺菌/殺真菌濃度(MIC、MBC、MFC)決定に対し、spot-plating技術による微量液体希釈法を用いて薬剤感受性試験を実施した。オイルおよびそのフェノール成分の殺菌および殺真菌活性の証明に、time-kill kinetics assayを実施した。外耳炎を有する犬から採材したメチシリン感受性Staphylococcus pseudintermedius(n = 10)、メチシリン耐性S. pseudintermedius(n = 10)、β溶血性連鎖球菌(n = 20)、緑膿菌(n = 20; 1つまたは2つの抗菌薬に耐性を持つ10の分離株を含む)、Proteus mirabilis(n = 20)、およびMalassezia pachydermatis(n = 20)を含む100の細菌および真菌分離株を本研究に供した。 結果: オレガノ油、タイム油、カルバクロールおよびチモールは、試験したすべての細菌および真菌分離株に対し抗菌活性を示した。 MIC背景: 多重耐药菌的抗菌治疗是一项全球均需面临的重要挑战,并且其经常使犬外耳炎复杂化。 假设/目的: 研究牛至油、百里香油及其主要酚类成分,对细菌和真菌引起的犬外耳炎的体外疗效。与精油相比,推测主要酚类成分具有更高的抗菌活性。 方法和材料: 使用肉汤微量稀释和点镀技术进行药敏试验,以确定最小抑菌、杀菌和杀真菌浓度(MIC、MBC和MFCs)。采用时间-杀菌动力试验以确定精油及其酚类成分的杀菌和杀真菌活性。共选取从犬外耳炎中分离的100株细菌和真菌菌株,包括甲氧西林敏感(n = 10)和甲氧西林耐药(n = 10)的假中间型葡萄球菌、β-溶血性链球菌(n = 20)、铜绿假单胞菌(n = 20;包括对一种或两种抗生素耐药的10个菌株)、奇异变形杆菌(n = 20)和厚皮马拉色菌(p = 20)。 结果: 牛至油、百里香油、香芹酚和百里香酚对所有检测的细菌和真菌菌株均有抗菌活性。对于革兰氏阳性菌和奇异变形杆菌,MICPatógenos multirresistentes representam um grande desafio global na terapia antimicrobiana e frequentemente complicam otites externas em cães. HIPÓTESE/OBJETIVOS: Investigou-se a eficácia in vitro do óleo de orégano, óleo de tomilho e seus principais componentes fenólicos contra isolados bacterianos e fúngicos associados com otite externa canina. A hipótese foi de que os principais componentes fenólicos teriam maior atividade antimicrobiana quando comparados com o óleo essencial relativo. MÉTODOS E MATERIAIS: Realizou-se teste de suscetibilidade a antimicrobianos utilizando a microdiluição em caldo com técnica de plaqueamento pontual para determinar a concentração inibitória mínima e as concentrações bactericidas/fungicidas (MICs, MBCs e MFCs). Um ensaio de cinética de tempo de morte foi realizado para confirmar a atividade bactericida e fungicida dos óleos e seus componentes fenólicos. Foram utilizados cem isolados bacterianos e fúngicos de cães com otite externa, incluindo Staphylococcus pseudintermedius suscetível à meticilina (n = 10), S. pseudintermedius resistente à meticilina (n = 10), Streptococcus spp β-hemolítico (n = 20), Pseudomonas aeruginosa (n = 20, incluindo 10 isolados resistentes a um ou dois antimicrobianos), Proteus mirabilis (n = 20) e Malassezia pachudermatis (n = 20).O óleo de orégano, óleo de tomilho, carvacrol e timol exibiram atividade antimicrobiana contra todos os isolados bacterianos e fúngicos testados. Os valores de MIC

The emergence and global spread of antimicrobial resistance among bacterial pathogens demand alternative strategies to treat life-threatening infections. Combination drugs and repurposing of old compounds with known safety profiles that are not currently used in human medicine can address the problem of multidrug-resistant infections and promote antimicrobial stewardship in veterinary medicine. In this study, the antimicrobial activity of robenidine alone or in combination with ethylenediaminetetraacetic acid (EDTA) or polymyxin B nonapeptide (PMBN) against Gram-negative bacterial pathogens, including those associated with canine otitis externa and human skin and soft tissue infection, was evaluated in vitro using microdilution susceptibility testing and the checkerboard method. Fractional inhibitory concentration indices (FICIs) and dose reduction indices (DRI) of the combinations against tested isolates were determined. Robenidine alone was bactericidal against Acinetobacter baumannii [minimum inhibitory concentrations (MIC) mode = 8 μg/ml] and Acinetobacter calcoaceticus (MIC mode = 2 μg/ml). Against Acinetobacter spp., an additivity/indifference of the combination of robenidine/EDTA (0.53 > FICIs > 1.06) and a synergistic effect of the combination of robenidine/PMBN (0.5 < FICI) were obtained. DRIs of robenidine were significantly increased in the presence of both EDTA and PMBN from 2- to 2048-fold. Robenidine exhibited antimicrobial activity against Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, in the presence of sub-inhibitory concentrations of either EDTA or PMBN. Robenidine also demonstrated potent antibacterial activity against multidrug-resistant Gram-positive pathogens and all Gram-negative pathogens isolated from cases of canine otitis externa in the presence of EDTA. Robenidine did not demonstrate antibiofilm activity against Gram-positive and Gram-negative bacteria. EDTA facilitated biofilm biomass degradation for both Gram-positives and Gram-negatives. The addition of robenidine to EDTA was not associated with any change in the effect on biofilm biomass degradation. The combination of robenidine with EDTA or PMBN has potential for further exploration and pharmaceutical development, such as incorporation into topical and otic formulations for animal and human use. Refereed/Peer-reviewed

An electrochemically activated solution (ECAS), generated by electrolysis of a dilute sodium chloride solution in a four-chamber electrolytic cell (Ecas4), was tested as a sanitising aerosol in eliminating bacteria from the environment of a weaning room vacated 24-48h earlier, at a continuous flow pig farm. An ultrasonic humidifier was used to fill the environment with a fog (droplets with diameters of 1–5 μm) containing 0.25 ppm of hypochlorous acid. The weaning room was fogged for 3 min at 30 min intervals during five hours of aerosol disinfection. An innovative sample treatment with propidium monoazide dye in conjunction with cyclonic air sampling was optimised and adapted for discerning live/dead bacteria in subsequent molecular quantification steps. Without fogging, total bacterial load ranged from 5.06 ± 0.04 to 5.75 ± 0.04 Log10 CFU/m3. After the first hour of fogging, a 78% total bacterial reduction was observed, which further increased to > 97% after the second hour, > 99.4% after the third and 99.8% after the fourth hour, finally resulting in a 99.99% reduction from the farm environment over five hours. Unlike the current formaldehyde spray disinfection protocol, which requires a long empty period because of its hazardous properties, this economically viable and environmentally friendly disinfection protocol may significantly lower downtime. Moreover, ECAS fogging can be easily adapted to a variety of applications, including the elimination of pathogens from livestock farm air environment for disease prevention, as well as decontamination after disease outbreaks. Refereed/Peer-reviewed

Increasing reports of multidrug-resistant bacterial infections in animals has created a need for novel antimicrobial agents that do not promote cross-resistance to critically important antimicrobial classes used in human medicine. In response to the recent emergence of antimicrobial resistance in several bovine mastitis pathogens, in vitro antimicrobial susceptibility was determined for four polyether ionophores (lasalocid, monensin, narasin and salinomycin) against Staphylococcus spp. and Streptococcus spp. isolated from clinical cases. In addition, erythrocyte haemolysis and WST-1 cell proliferation assays were used to assess in vitro mammalian cell cytotoxicity and biofilm susceptibility testing was performed using the minimum biofilm eradication concentration(MBEC™) biofilm assay. Lasalocid, monensin, narasin and salinomycin exhibited bacteriostatic antimicrobial activity against all pathogens tested, including methicillin-resistant staphylococci, with MIC90 values

There are very few articles in the literature describing continuous models of bacterial infections that mimic disease pathogenesis in humans and animals without using separate cohorts of animals at each stage of disease. In this work, we developed bioluminescent mouse models of partial-thickness scald wound infection and sepsis that mimic disease pathogenesis in humans and animals using a recombinant luciferase-expressing Staphylococcus aureus strain (Xen29). Two days post-scald wound infection, mice were treated twice daily with a 2% topical mupirocin ointment for 7 days. For sepsis experiments, mice were treated intraperitoneally with 6 mg/kg daptomycin 2 h and 6 h post-infection and time to moribund monitored for 72 h. Consistent bacterial burden data were obtained from individual mice by regular photon intensity quantification on a Xenogen IVIS Lumina XRMS Series III biophotonic imaging system, with concomitant significant reduction in photon intensities in drug-treated mice. Post-mortem histopathological examination of wounds and bacterial counts in blood correlated closely with disease severity and total flux obtained from Xen29. The bioluminescent murine models provide a refinement to existing techniques of multiple bacterial enumeration during disease pathogenesis and promote animal usage reduction. The models also provide an efficient and information-rich platform for preclinical efficacy evaluation of new drug classes for treating acute and chronic human and animal bacterial infections. Refereed/Peer-reviewed

Methicillin-resistant coagulase-negative staphylococci (MRCoNS) have recently emerged as a significant cause of bovine mastitis worldwide. Here we describe the isolation of MRCoNS from cases of bovine mastitis from a single dairy farm in Australia. Fourteen CoNS isolates were identified as MRCoNS on the basis of having an oxacillin MIC of ≥0.5 μg/mL. The isolates were speciated as S. chromogenes (n = 1) S. fleurettii (n = 1), S. haemolyticus (n = 2), S. sciuri (n = 5), S. simulans (n = 1) S. succinus (n = 2) and S. xylosus (n = 2). Five of the isolates (S. fleuretti, S. haemolyticus S. sciuri and two S. succinus) were mecA-positive. We also detected a previously described S. sciuri mecA homolog in four oxacillin-resistant S. sciuri isolates. The remainder of the putative MRCoNS did not contain any mecA-related resistance determinants in their genomes. Comparative genomic analysis of three previously published S. sciuri isolates, from humans, a squirrel and a cereal crop (rice), and a representative isolate from our study demonstrated clustering and a high degree of genetic homogeneity ( > 95%), suggesting S. sciuri has low host specificity. In conclusion, CoNS, in particular S. sciuri, may act as a reservoir for SCCmec elements that can easily be spread between different host species by direct cross-infection. Refereed/Peer-reviewed

Made available in DSpace on 2018-11-26T17:40:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-09-05 Australian Research Council (ARC) Neoculi Pty Ltd National Health and Medical Research Council of Australia (NHMRC) University of South Australia Sansom Institute of Health Research Micromyx LLC The spread of multidrug resistance among bacterial pathogens poses a serious threat to public health worldwide. Recent approaches towards combating antimicrobial resistance include repurposing old compounds with known safety and development pathways as new antibacterial classes with novel mechanisms of action. Here we show that an analog of the anticoccidial drug robenidine (4,6-bis(2-((E)-4-methylbenzylidene)hydrazinyl)pyrimidin-2-amine; NCL195) displays potent bactericidal activity against Streptococcus pneumoniae and Staphylococcus aureus by disrupting the cell membrane potential. NCL195 was less cytotoxic to mammalian cell lines than the parent compound, showed low metabolic degradation rates by human and mouse liver microsomes, and exhibited high plasma concentration and low plasma clearance rates in mice. NCL195 was bactericidal against Acinetobacter spp and Neisseria meningitidis and also demonstrated potent activity against A. baumannii, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Enterobacter spp. in the presence of sub-inhibitory concentrations of ethylenediaminetetraacetic acid (EDTA) and polymyxin B. These findings demonstrate that NCL195 represents a new chemical lead for further medicinal chemistry and pharmaceutical development to enhance potency, solubility and selectivity against serious bacterial pathogens. Univ Adelaide, Sch Anim & Vet Sci, Australian Ctr Antimicrobial Resistance Ecol, Roseworthy, SA, Australia Univ Newcastle, Sch Environm & Life Sci, Chem, Callaghan, NSW, Australia Univ Adelaide, Sch Anim & Vet Sci, Roseworthy, SA, Australia Neoculi Pty Ltd, Burwood, Vic, Australia Univ South Australia, Sansom Inst Hlth Res, Sch Pharm & Med Sci, Ctr Pharmaceut Innovat & Dev, Adelaide, SA, Australia Univ South Australia, Sansom Inst Hlth Res, Sch Pharm & Med Sci, Adelaide, SA, Australia Monash Univ, Monash Inst Pharmaceut Sci, Ctr Drug Candidate Optimisat, Parkville, Vic, Australia Sao Paulo State Univ, Sch Pharmaceut Sci, Dept Drugs & Med, Sao Paulo, Brazil Fiona Stanley Hosp, Path West Lab Med WA, Dept Microbiol, Murdoch, WA, Australia Murdoch Univ, Sch Vet & Life Sci, Murdoch, WA, Australia Micromyx LLC, Kalamazoo, MI USA Univ Adelaide, Sch Biol Sci, Dept Mol & Cellular Biol, Adelaide, SA, Australia Univ Adelaide, Sch Biol Sci, Dept Mol & Cellular Biol, Res Ctr Infect Dis, Adelaide, SA, Australia Sao Paulo State Univ, Sch Pharmaceut Sci, Dept Drugs & Med, Sao Paulo, Brazil Australian Research Council (ARC): LP110200770 National Health and Medical Research Council of Australia (NHMRC): 565526 National Health and Medical Research Council of Australia (NHMRC): 627142

Robenidine, 1 (2,2′-bis[(4-chlorophenyl)methylene]carbonimidic dihydrazide), was active against MRSA and VRE with MIC’s of 8.1 and 4.7 μM, respectively. SAR revealed tolerance for 4-Cl isosteres with 4-F (8), 3-F (9), 3-CH3 (22), and 4-C(CH3)3 (27) (23.7–71 μM) and with 3-Cl (3), 4-CH3 (21), and 4-CH(CH3)2 (26) (8.1–13.0 μM). Imine carbon alkylation identified a methyl/ethyl binding pocket that also accommodated a CH2OH moiety (75; 2,2′-bis[1-(4-chlorophenyl)-2-hydroxyethylidene]carbonimidic dihydrazide). Analogues 1, 27 (2,2′-bis{[4-(1,1-dimethylethyl)phenyl]methylene}carbonimidic dihydrazide), and 69 (2,2′-bis[1-(4-chlorophenyl)ethylidene]carbonimidic dihydrazide hydrochloride) were active against 24 clinical MRSA and MSSA isolates. No dose-limiting cytotoxicity at ≥2× MIC or hemolysis at ≥8× MIC was observed. Polymyxin B addition engendered Escherichia coli and Pseudomonas aeruginosa Gram-negative activity MIC’s of 4.2–21.6 μM. 1 and 75 displayed excellent microsomal stability, intrinsic clearance, and hepatic extraction ratios with T1/2 > 247 min, CLint < 7 μL/min/mg protein, and EH < 0.22 in both human and mouse liposomes for 1 and in human liposomes for 75. Refereed/Peer-reviewed

Developing a reliable mastitis challenge infection model is required to test new intramammary antimicrobial preparations, other novel bovine mastitis treatments, and study mastitis pathogenesis. Three treatment groups of Holstein Friesian cows in active lactation were administered two doses (104 and 106 cfu/quarter) on a single occasion with one of the three Streptococcus uberis strains (BFR6019, MFF1283 and SA002) suspended in 5 ml of sterile PBS, administered via intramammary inoculation immediately after milking. All quarters that were challenged with S. uberis strains MLF1283 and BFR6019 showed clinical signs of mastitis on day 1 and 2 after the challenge. Strain SA002 had a lower rate of inducing clinical mastitis which was detected later than day 3 after the challenge. We successfully developed a rapid and reliable model for inducing experimental S. uberis mastitis with 100% success rate in cows in active lactation. On the basis of the correlation results between strains, RAPD fingerprinting results, clinical findings, and a 100% success rate of mastitis induction for low and high doses S. uberis strains MLF1283 and BFR6019, strain virulence seems to be a more important effect than challenge dose in induction of clinical mastitis following experimental challenge.