In vitro antimicrobial activity of narasin and monensin in combination with adjuvants against pathogens associated with canine otitis externa.

BACKGROUND: The emergence of antimicrobial resistance represents a serious human and animal health risk. Good antimicrobial stewardship is essential to prolong the lifespan of existing antibiotics, and new strategies are required to combat infections in man and animals. HYPOTHESIS/OBJECTIVES: To determine the in vitro interaction of ionophores (narasin or monensin) with antimicrobial adjuvants (N-acetylcysteine (NAC), Tris-EDTA or disodium EDTA) against bacterial strains representing pathogens associated with canine otitis externa (OE). ANIMAL/ISOLATES: American Type Culture Collection (ATCC) strains Staphylococcus aureus 29213, Pseudomonas aeruginosa 27853 and P. aeruginosa biofilm producer PAO1, and a clinical isolate of Proteus mirabilis from a case of canine OE were tested. METHODS AND MATERIALS: A 2D microdilution checkerboard method was used, allowing calculation of fractional inhibitory concentration index (FICI), dose reduction index (DRI) and plotting of isobolograms. RESULTS: The combination of narasin with either Tris-EDTA or disodium EDTA produced additive effects (FICI = 0.75) against P. aeruginosa ATCC 27853 and P. aeruginosa biofilm producer ATCC PAO1. An additive effect (FICI = 0.53-0.75) was found against S. aureus ATCC 29213 when narasin or monensin were combined with NAC. The highest DRI (32-fold) was found with monensin/NAC where the MIC of monensin was reduced from 4 to 0.125 µg/mL. CONCLUSIONS AND CLINICAL IMPORTANCE: The combination of narasin with Tris-EDTA or disodium EDTA is a promising strategy to inhibit the intrinsic resistance elements of Gram-negative bacteria. These novel combinations potentially could be useful as a multimodal approach to treat mixed infections in canine OE.

Allicin prevents the formation of Proteus-induced urinary crystals and the blockage of catheter in a bladder model in vitro.

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, Mg2+ and Ca2+ 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, Mg2+ and Ca2+ 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 Mg2+ and Ca2+ deposition in a dose-dependent manner.

Antimicrobial activity of thyme oil, oregano oil, thymol and carvacrol against sensitive and resistant microbial isolates from dogs with otitis externa.

BACKGROUND: Multidrug-resistant pathogens present a major global challenge in antimicrobial therapy and frequently complicate otitis externa in dogs. HYPOTHESIS/OBJECTIVES: 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. METHODS AND MATERIALS: 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), ß-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. RESULTS: Oregano oil, thyme oil, carvacrol and thymol exhibited antibacterial activity against all bacterial and fungal isolates tested. MIC90 values ranged from 0.015 to 0.03% (146-292 µg/mL) for the Gram-positive bacteria and P. mirabilis. For P. aeruginosa and M. pachydermatis, MIC90 values ranged from 0.09 to 0.25% (800-2,292 µg/mL). CONCLUSIONS AND CLINICAL SIGNIFICANCE: Oregano 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.

In vitro antimicrobial activity of seven adjuvants against common pathogens associated with canine otitis externa.

BACKGROUND: An antibiotic adjuvant is a chemical substance used to modify or augment the effectiveness of primary antimicrobial agents against drug-resistant micro-organisms. Its use provides an alternative approach to address the global issue of antimicrobial resistance and enhance antimicrobial stewardship. HYPOTHESIS/OBJECTIVES: To determine the antimicrobial activity of a panel of potential antimicrobial adjuvants against common pathogens associated with canine otitis externa (OE). ANIMALS/ISOLATES: A number of type strains and clinical isolates (n = 110) from canine OE were tested including Staphylococcus pseudintermedius, ß-haemolytic Streptococcus spp., Pseudomonas aeruginosa, Proteus mirabilis and Malassezia pachydermatis. METHODS AND MATERIALS: Antimicrobial activities of monolaurin, monocaprin, N-acetylcysteine (NAC), polymyxin B nonapeptide, Tris-EDTA, Tris-HCL and disodium EDTA were tested using microdilution methodology according to CLSI guidelines. RESULTS: N-acetylcysteine, Tris-EDTA and disodium EDTA had antimicrobial activity against both type strains and otic pathogens. The other adjuvants tested had limited to no efficacy. NAC had a minimal inhibitory concentration (MIC) range of 2,500-10,000 µg/mL for the various organisms. Pseudomonas aeruginosa isolates were eight times more susceptible to disodium EDTA in the presence of Tris-HCL in comparison to disodium EDTA alone. Malassezia pachydermatis isolates were most susceptible to Tris-EDTA (MIC90  = 190/60 µg/mL) and disodium EDTA (MIC90  = 120 µg/mL). CONCLUSIONS AND CLINICAL RELEVANCE: N-acetylcysteine, Tris-EDTA and disodium EDTA have intrinsic antimicrobial activity and represent promising adjuvants that could be used to enhance the efficacy of existing antibiotics against Gram-negative and multidrug-resistant bacterial infections. These agents could be combined with other antimicrobial agents in a multimodal approach for mixed ear infections in dogs.

In vitro antimicrobial activity of narasin against common clinical isolates associated with canine otitis externa.

BACKGROUND: Antimicrobial resistance and antimicrobial stewardship are of ever-increasing importance in veterinary medicine. Re-purposing of old drugs that are not used in human medicine is one approach that addresses the emergence of multidrug resistance in canine skin and ear infections, and can reduce the use of critically important human antibiotic classes. HYPOTHESIS/OBJECTIVES: To determine the antimicrobial activity of narasin, a polyether ionophore conventionally used as a rumen modifier and anticoccidial agent in production animals, against common clinical isolates of canine otitis externa (OE). ANIMALS/ISOLATES: Clinical isolates (n = 110) from canine OE were tested, including 17 meticillin-susceptible Staphylococcus pseudintermedius (MSSP), 13 multidrug-resistant Staphylococcus pseudintermedius (MDRSP), and 20 each of ß-haemolytic Streptococcus spp., Pseudomonas aeruginosa, Proteus mirabilis and Malassezia pachydermatis. METHODS: Bacterial and yeast isolates were subcultured, suspended in broth and inoculated into 96-well plates. Organisms were tested against concentrations of narasin ranging from 0.03 to 128 µg/mL. Minimal inhibitory concentrations (MICs) were determined after overnight incubation. RESULTS: Narasin MICs for staphylococcal and streptococcal isolates ranged from 0.06 to 0.25 µg/mL; MIC50 and MIC90 values for both organisms were 0.125 µg/mL. No MICs were achieved for Pseudomonas or Proteus isolates. There was a weak antifungal effect against M. pachydermatis isolates (MIC 32 to >128 µg/mL). CONCLUSIONS AND CLINICAL RELEVANCE: Narasin was effective against Gram-positive bacteria and had antifungal activity at higher concentrations against M. pachydermatis. However, the lack of Gram-negative activity would prevent its use as a sole antimicrobial agent in cases of canine OE.

Repurposing Ionophores as novel antimicrobial agents for the treatment of bovine mastitis caused by Gram-positive pathogens.

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 <16 µg/ml. Narasin and monensin displayed the least toxicity against mammalian cell lines and all compounds significantly reduced viable cell numbers in a Staphylococcus aureus biofilm. Based on in vitro characterization, all four ionophores offer potentially novel treatments against bovine mastitis but in vivo studies will be essential to determine whether acceptable safety and efficacy is present following intramammary administration.

Evaluation of effects of Mycoplasma mastitis on milk composition in dairy cattle from South Australia.

BACKGROUND: Mycoplasma mastitis is increasingly posing significant impact on dairy industry. Although the effects of major conventional mastitis pathogens on milk components has been widely addressed in the literature, limited data on the effects of different Mycoplasma and Acholeplasma spp. on milk quality and quantity is available. The aim of this study was to determine the casual relationship of Mycoplasma spp. and A. laidlawii to mastitis and compare them to subclinical mastitis caused by conventional mastitis pathogens from a single dairy herd in South Australia; Mycoplasma spp. and A. laidlawii were detected using PCR applied directly to milk samples. The herd had mastitis problem with high somatic cell count and low response rate to conventional antimicrobial therapy. A total of 288 cow-level milk samples were collected aseptically and used in this study. RESULTS: Conventional culture showed a predominance of coagulase-negative staphylococci, followed by coagulase-positive staphylococci, Streptococcus spp., Enterococcus spp., E. coli, and Klebsiella spp. PCR results showed a high prevalence of mycoplasmas (76.7%), including A. laidlawii (10.8%), M. bovis (6.2%), M. bovirhinis (5.6%), M. arginini (2%), and (52.1%) of cows were co-infected with two or more Mycoplasma and Acholeplasma species. Mycoplasma co-infection significantly increased somatic cell counts (SCC) similar to conventional mastitis pathogens and compared to non-infected cows with 389.3, 550.3 and 67.3 respectively; and decreased the milk yield with 29.0, 29.9 and 34.4 l, respectively. Mycoplasma co-infection caused significant increase in protein percentage, and significant decrease in fat percentage and total milk solids, similar to other conventional mastitis pathogens. In contrast, changes in milk composition and yield caused by various individual Mycoplasma species were non-significant. CONCLUSIONS: Mycoplasma mastitis had on-farm economic consequences similar to common conventional mastitis pathogens. Results of our study indicate that co-infection Mycoplasma mastitis caused similar effect on milk composition to other mastitis pathogens and we hope these findings raise the awareness of the importance of their detection on routine diagnostic panels.

Efficacy evaluation of a new water sanitizer for increasing the shelf life of Southern Australian King George Whiting and Tasmanian Atlantic Salmon fillets.

The bacterial species and specific spoilage organisms associated with the Southern Australian King George Whiting (KGW) and Tasmanian Atlantic Salmon (TAS), and the efficacy of a HOCl-containing water-based sanitization product (Electro-Chemically Activated Solution, by ECAS4) in extending the shelf life of KGW and TAS fillets were evaluated. Fillets were washed with an ECAS4 solution containing either 45 ppm or 150 ppm of free chlorine and bacterial species enumerated on selective and non-selective media, followed by identification of pure isolates by 16 S rRNA gene sequencing. The dominant spoilage microbiota in KGW and TAS fillets stored at 4 ± 1 °C were Pseudomonas spp. and Shewanella spp. At either concentration, ECAS4 significantly reduced total bacterial load and specific spoilage organisms on KGW and TAS fillets (approx. 1-2 log colony-forming units) during storage and significantly extended the shelf life of the fillets by 2 and 4 days, respectively. The significant increase in shelf life and quality of fillets was corroborated by raw and cooked sensory evaluation. ECAS4 sanitization could have a significant impact on the overall food industry, translating into health and economic benefits through reduction of food spoilage bacteria and potentially, foodborne pathogens without many of the disadvantages of currently approved biocides.

Development of an improved Streptococcus uberis experimental mastitis challenge model using different doses and strains in lactating dairy cows.

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 (10(4) and 10(6) 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.

In vitro efficacy of cefovecin against anaerobic bacteria isolated from subgingival plaque of dogs and cats with periodontal disease.

Periodontal disease is a common disease of dogs and cats often requiring antimicrobial treatment as an adjunct to mechanical debridement. However, correct compliance with oral antimicrobial therapy in companion animals is often difficult. Cefovecin is a recently introduced veterinary cephalosporin that has demonstrated prolonged concentrations in extracellular fluid, allowing for dosing intervals of up to 14 days. Subgingival samples were collected from the oral cavity of 29 dogs and eight cats exhibiting grade 2 or grade 3 periodontal disease. Samples were cultivated on Wilkin Chalgrens agar and incubated in an anaerobic chamber for seven days. Selected anaerobic bacteria were isolated and identified to species level using 16S rRNA gene sequence analysis. Minimum inhibitory concentrations were determined for cefovecin and six additional antimicrobials using the agar dilution methodology recommended by the Clinical and Laboratory Standards Institute. The 65 clinical isolates were identified as Porphyromonas gulae (n = 45), Porphyromonas crevioricanis (n = 12), Porphyromonas macacae (n = 1), Porphyromonas cangingivalis (n = 1) Fusobacterium nucleatum (n = 2), Fusobacterium russii (n = 1) and Solobacterium moorei (n = 3). This is the first report of S. moorei being isolated from companion animals with periodontal disease. All isolates were highly susceptible to cefovecin, with a MIC90 of ≤0.125 µg/ml. Conversely, different resistance rates to ampicillin, amoxicillin and erythromycin between isolates were detected. Cefovecin is thus shown to be effective in vitro against anaerobic bacteria isolated from dogs and cats with periodontal disease.

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 [...].

Advances in understanding the specificity function of transporters by machine learning.

Understanding the underlying molecular mechanism of transporter activity is one of the major discussions in structural biology. A transporter can exclusively transport one ion (specific transporter) or multiple ions (general transporter). This study compared categorical and numerical features of general and specific calcium transporters using machine learning and attribute weighting models. To this end, 444 protein features, such as the frequency of dipeptides, organism, and subcellular location, were extracted for general (n = 103) and specific calcium transporters (n = 238). Aliphatic index, subcellular location, organism, Ile-Leu frequency, Glycine frequency, hydrophobic frequency, and specific dipeptides such as Ile-Leu, Phe-Val, and Tyr-Gln were the key features in differentiating general from specific calcium transporters. Calcium transporters in the cell outer membranes were specific, while the inner ones were general; additionally, when the hydrophobic frequency or Aliphatic index is increased, the calcium transporter act as a general transporter. Random Forest with accuracy criterion showed the highest accuracy (88.88% ±5.75%) and high AUC (0.964 ± 0.020), based on 5-fold cross-validation. Decision Tree with accuracy criterion was able to predict the specificity of calcium transporter irrespective of the organism and subcellular location. This study demonstrates the precise classification of transporter function based on sequence-derived physicochemical features.

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.

Developing an in vitro method for Eimeria tenella attachment to its preferred and non-preferred intestinal sites.

A frozen section method utilising chicken intestinal tissue was developed to study the Eimeria tenella attachment ex vivo. In order to examine Eimeria-epithelial cell attachment, 10(5) E. tenella sporozoites were incubated with each caecal frozen section (6, 10 and 14 microm) for 1h in 5% CO2 incubator at 41 degrees C. E. tenella sporozoites attached successfully to enterocytes in 14 microm thick of caecal sections. Sporozoite attachment to caecal sections was shown to be dependent on the number of parasites added. To evaluate the method, E. tenella sporozoites were incubated to its preferred (caecum) and non-preferred (duodenum and jejunum) intestinal sites. The number of sporozoites attached to the caecal enterocytes was significantly greater (P<0.0001) in comparison with the limited number of sporozoites attached to enterocytes of non-preferred intestinal sites. This method was shown to be able to reveal differences in binding capability and allows for comparison of intestinal site attachment.

Decontamination of aerosolised bacteria from a pig farm environment using a pH neutral electrochemically activated solution (Ecas4 anolyte).

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.

Discovery of 4,6-bis(2-((E)-benzylidene)hydrazinyl)pyrimidin-2-Amine with Antibiotic Activity.

Robenidine (E)-N'-((E)-1-(4-chlorophenyl)ethylidene)-2-(1-(4-chlorophenyl)ethylidene)hydrazine-1-carboximidhydrazide displays methicillin-resistant Staphyoccoccus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) MICs of 2 µg mL-1. Herein we describe the structure-activity relationship development of a novel series of guanidine to 2-aminopyrimidine isosteres that ameliorate the low levels of mammalian cytotoxicity in the lead compound while retaining good antibiotic activity. Removal of the 2-NH2 pyrimidine moiety renders these analogues inactive. Introduction of a central 2-NH2 triazine moiety saw a 10-fold activity reduction. Phenyl to cyclohexyl isosteres were inactive. The 4-BrPh and 4-CH3Ph with MIC values of 2 and 4 µg mL-1, against MRSA and VRE respectively, are promising candidates for future development.

In vitro Antimicrobial Activity of Robenidine, Ethylenediaminetetraacetic Acid and Polymyxin B Nonapeptide Against Important Human and Veterinary Pathogens.

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.

Evaluation of three cryoprotectants used with bovine milk affected with Mycoplasma bovis in different freezing conditions.

OBJECTIVES: Currently, there is no consensus protocols regarding the combination of glycerol (GLY), gelatin or foetal bovine serum (FBS) with dimethyl sulphoxide (DMSO) as cryoprotectants for Mycoplasma bovis in bovine milk samples. This study aimed to compare different cryopreservation compounds and storage temperatures for M. bovis. RESULTS: There were significant differences in the survival of M. bovis on different media. Differences were also observed between different storage conditions. All additives improved the survival of M. bovis in comparison to control (CON). The combination of GLY and DMSO was shown to be significantly different to CON with 57.1% (95% CI = 21.43-133.34) and 19.1% (95% CI = 11.73-60.27), respectively at week 16, and its use should be encouraged as a cryoprotectant for M. bovis at - 20 and - 80 °C. GEL/DMSO showed the highest survival rate for M. bovis with 57.14% (95% CI = 21.43-133.34) at 4 °C in comparison with CON 14.29% (95% CI = 9.60-50.39). FBS/DMSO showed the highest survival rate for the short-term preservation similarly to other additives. The evaluated cryopreservative compounds would improve survivability of M. bovis in milk for both transport and long-term storage. Hence, it is recommended to use the mentioned methods for routine transportation or storage purposes for suspicious M. bovis milk samples.

In vitro antimicrobial activity of monensin against common clinical isolates associated with canine otitis externa.

Antimicrobial resistance and antimicrobial stewardship are of ever-increasing importance in veterinary medicine. Multidrug-resistant infections of the canine skin and ear continue to emerge, but the use of antibiotic classes of critical importance to human medicine may not represent good antimicrobial stewardship. Repurposing of old drugs that are not used in human medicine is one approach that addresses both these issues. In this study, the minimal inhibitory concentration (MIC) of monensin for 111 bacterial and yeast canine otitis isolates was determined using microdilution methodology according to Clinical Laboratory Standards Institute (CLSI) guidelines. Monensin was effective against all Gram-positive bacteria including the multidrug-resistant staphylococcal strains with MICs ranging from 1 to 4 µg/ml, but lacked antimicrobial activity against Gram-negative bacteria and yeast isolates. Monensin has potential to be incorporated as one of the main components in an otic formulation.