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.

Antimicrobial resistance in clinical Escherichia coli isolated from companion animals in Australia.

Multidrug-resistant (MDR) Escherichia coli have become a major public health concern to both humans and animal health. While the frequency of antimicrobial resistance (AMR) in clinical E. coli is monitored regularly in human medicine, current frequency of AMR in companion animals remains unknown in Australia. In this study we conducted antimicrobial susceptibility testing (AST) and where possible, determined potential risk factors for MDR infection among 883 clinical Escherichia coli isolated from dogs (n=514), cats (n=341) and horses (n=28). AST was undertaken for 15 antimicrobial agents according to the Clinical Laboratory Standards Institute (CLSI) guidelines and interpreted using epidemiological cut-off values (ECOFFs) as well as CLSI veterinary and human clinical breakpoints. The AST revealed complete absence of resistance to carbapenems while resistance to amikacin was observed at a low level in isolates from dogs (1.6%) and cats (1.5%) compared to horses (10.7%). Among dog isolates, resistance to fluoroquinolones ranged from 9.1%-9.3% whereas among cat isolates, it ranged from 3.2%-5%. Among dog isolates, the proportion showing a 3rd generation cephalosporin (3GC) non-wild type phenotype was significantly higher (P<0.05) in skin and soft tissue infection (SSTI, n=122) isolates (17.2%-20.5%) compared to urinary tract infection (UTI, n=392) isolates (9.9%-10.2%). The frequency of multidrug resistance was 18.1%, 11.7% and 42.9% in dog, cat and horse isolates, respectively. Risk factor analysis revealed that MDR E. coli isolated from UTI were positively associated with chronicity of infection and previous antimicrobial treatment. Dogs and cats with chronic UTI that had been previously treated with antimicrobials were eight times and six times more likely to be infected with MDR E. coli compared to dogs and cats with non-chronic UTI, and no history of antimicrobial treatment, respectively. This study revealed that pre-existing disease condition and prior antimicrobial use were the major risks associated with UTI with MDR E. coli in companion animals.

Robenidine Analogues as Gram-Positive Antibacterial Agents.

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.

Survival of Pseudomonas aeruginosa in modified Romanowsky staining solutions.

BACKGROUND: Anecdotal reports suggest that rapid staining solutions can become contaminated with micro-organisms, especially Pseudomonas aeruginosa. HYPOTHESIS/OBJECTIVES: To determine whether inoculation of rapid Romanowsky-type stains with P. aeruginosa results in viable bacterial contamination, which could lead to cross-contamination of slides during cytological staining. METHODS: Pseudomonas aeruginosa was inoculated into clean and organically contaminated staining solutions (fixative, eosin and methylene blue) and positive (broth) and negative (bleach) control solutions. Subsequent viability and survival were detected by measuring colony-forming units per millilitre at various time points up to 2 weeks. Each sample was stained and microscopically examined to determine whether bacteria were visible. RESULTS: No bacteria could be cultured at any time point from the bleach or fixative solution. In clean eosin and methylene blue staining solutions, viable bacteria were recovered for up to 1 h, but by 24 h all bacteria were dead. In staining solutions contaminated with hair and dead skin cells, bacteria survived in methylene blue for up to 1 h, and viable bacteria persisted in the eosin stain for 2 weeks. In solutions containing viable organisms, the bacteria could be observed by microscopic examination; no bacteria were visible when the solutions contained no viable organisms. CONCLUSIONS AND CLINICAL IMPORTANCE: Pseudomonas aeruginosa can survive in commonly used staining solutions for variable periods of time, but is unable to proliferate. Although theoretically this might complicate cytological interpretation and subsequent diagnosis, the likelihood of this in clinical practice appears remote when the correct staining technique is used.

Gas chromatography-mass spectrometry-based metabolite profiling of Salmonella enterica serovar Typhimurium differentiates between biofilm and planktonic phenotypes.

The aim of this study was to utilize gas chromatography coupled with mass spectrometry (GC-MS) to compare and identify patterns of biochemical change between Salmonella cells grown in planktonic and biofilm phases and Salmonella biofilms of different ages. Our results showed a clear separation between planktonic and biofilm modes of growth. The majority of metabolites contributing to variance between planktonic and biofilm supernatants were identified as amino acids, including alanine, glutamic acid, glycine, and ornithine. Metabolites contributing to variance in intracellular profiles were identified as succinic acid, putrescine, pyroglutamic acid, and N-acetylglutamic acid. Principal-component analysis revealed no significant differences between the various ages of intracellular profiles, which would otherwise allow differentiation of biofilm cells on the basis of age. A shifting pattern across the score plot was illustrated when analyzing extracellular metabolites sampled from different days of biofilm growth, and amino acids were again identified as the metabolites contributing most to variance. An understanding of biofilm-specific metabolic responses to perturbations, especially antibiotics, can lead to the identification of novel drug targets and potential therapies for combating biofilm-associated diseases. We concluded that under the conditions of this study, GC-MS can be successfully applied as a high-throughput technique for "bottom-up" metabolomic biofilm research.

Development of intramammary delivery systems containing lasalocid for the treatment of bovine mastitis: impact of solubility improvement on safety, efficacy, and milk distribution in dairy cattle.

BACKGROUND: Mastitis is a major disease of dairy cattle. Given the recent emergence of methicillin-resistant Staphylococcus aureus as a cause of bovine mastitis, new intramammary (IMA) treatments are urgently required. Lasalocid, a member of the polyether ionophore class of antimicrobial agents, has not been previously administered to cows by the IMA route and has favorable characteristics for development as a mastitis treatment. This study aimed to develop an IMA drug delivery system (IMDS) of lasalocid for the treatment of bovine mastitis. METHODS: Minimum inhibitory concentrations (MICs) were determined applying the procedures recommended by the Clinical and Laboratory Standards Institute. Solid dispersions (SDs) of lasalocid were prepared and characterized using differential scanning calorimetry and Fourier transform infrared spectroscopy. IMDSs containing lasalocid of micronized, nano-sized, or as SD form were tested for their IMA safety in cows. Therapeutic efficacy of lasalocid IMDSs was tested in a bovine model involving experimental IMA challenge with the mastitis pathogen Streptococcus uberis. RESULTS: Lasalocid demonstrated antimicrobial activity against the major Gram-positive mastitis pathogens including S. aureus (MIC range 0.5-8 µg/mL). The solubility test confirmed limited, ion-strength-dependent water solubility of lasalocid. A kinetic solubility study showed that SDs effectively enhanced water solubility of lasalocid (21-35-fold). Polyvinylpyrrolidone (PVP)-lasalocid SD caused minimum mammary irritation in treated cows and exhibited faster distribution in milk than either nano or microsized lasalocid. IMDSs with PVP-lasalocid SD provided effective treatment with a higher mastitis clinical and microbiological cure rate (66.7%) compared to cloxacillin (62.5%). CONCLUSION: Lasalocid SD IMDS provided high cure rates and effectiveness in treating bovine mastitis with acceptable safety in treated cows.

Carbapenemase-producing bacteria in companion animals: a public health concern on the horizon.

Clinical infections attributed to carbapenemase-producing bacteria are a pressing public health concern owing to limited therapeutic options and linked antimicrobial resistance. In recent years, studies have reported the emergence and spread of carbapenemase-producing Enterobacteriaceae and their public health impact. This has been closely followed by the global dissemination of highly resistant and virulent zooanthroponotic extraintestinal pathogenic Escherichia coli (ExPEC) ST131 clones. It has also been hypothesized that companion animals may act as a reservoir for Gram-negative multidrug-resistant pathogens in the community. Two recent reports have documented the emergence of carbapenemase-producing bacteria in companion animals. This phenomenon is of great concern because of the close contact between humans and their pets, and the potential for cross-species transmission. This scenario suggests a role for multifaceted control of Gram-negative multidrug-resistant infections in companion animals. This short article addresses this issue and identifies steps that could facilitate this process.

Comparative susceptibility of Salmonella Typhimurium biofilms of different ages to disinfectants.

There is a general consensus that with increasing age a biofilm shows increased resistance to antimicrobials. In this study the susceptibility of 3-, 5- and 7-day-old Salmonella enterica serovar Typhimurium biofilms to disinfectants was evaluated. It was hypothesized that 7-day-old biofilms would be more resistant to disinfectants compared to 3- and 5-day-old biofilms. Biofilms were formed using the MBEC™ system and treated with six chemical disinfectants for 1 and 5 min. Four disinfectants at the highest concentration available showed 100% reduction in viable cells from all ages of biofilms after exposure for 5 min, and ethanol at 70% v/v was the least effective against biofilms, followed by chlorhexidine gluconate (CG). At the recommended user concentrations, only sodium hypochlorite showed 100% reduction in viable cells from all ages of biofilms. Benzalkonium chloride and CG were the least effective against biofilms, followed by quaternary ammonium compound which only showed 100% reduction in viable cells from 5-day-old biofilms. Overall, the results from this study do not display enhanced resistance in 7-day-old biofilms compared to 3- and 5-day-old biofilms. It is concluded that under the conditions of this study, the age of biofilm did not contribute to resistance towards disinfectants. Rather, the concentration of disinfectant and an increased contact time were both shown to play a role in successful sanitization.