Genomic epidemiology of third-generation cephalosporin-resistant Escherichia coli from Argentinian pig and dairy farms reveals animal-specific patterns of co-resistance and resistance mechanisms.

Control measures are being introduced globally to reduce the prevalence of antibiotic resistance (ABR) in bacteria on farms. However, little is known about the current prevalence and molecular ecology of ABR in bacterial species with the potential to be key opportunistic human pathogens, such as Escherichia coli, on South American farms. Working with 30 dairy cattle farms and 40 pig farms across two provinces in central-eastern Argentina, we report a comprehensive genomic analysis of third-generation cephalosporin-resistant (3GC-R) E. coli, which were recovered from 34.8% (cattle) and 47.8% (pigs) of samples from fecally contaminated sites. Phylogenetic analysis revealed substantial diversity suggestive of long-term horizontal and vertical transmission of 3GC-R mechanisms. CTX-M-15 and CTX-M-2 were more often produced by isolates from dairy farms, while CTX-M-8 and CMY-2 and co-carriage of amoxicillin/clavulanate resistance and florfenicol resistance were more common in isolates from pig farms. This suggests different selective pressures for antibiotic use in these two animal types. We identified the ß-lactamase gene blaROB, which has previously only been reported in the family Pasteurellaceae, in 3GC-R E. coli. blaROB was found alongside a novel florfenicol resistance gene, ydhC, also mobilized from a pig pathogen as part of a new composite transposon. As the first comprehensive genomic survey of 3GC-R E. coli in Argentina, these data set a baseline from which to measure the effects of interventions aimed at reducing on-farm ABR and provide an opportunity to investigate the zoonotic transmission of resistant bacteria in this region. IMPORTANCE: Little is known about the ecology of critically important antibiotic resistance among bacteria with the potential to be opportunistic human pathogens (e.g., Escherichia coli) on South American farms. By studying 70 pig and dairy cattle farms in central-eastern Argentina, we identified that third-generation cephalosporin resistance (3GC-R) in E. coli was mediated by mechanisms seen more often in certain species and that 3GC-R pig E. coli were more likely to be co-resistant to florfenicol and amoxicillin/clavulanate. This suggests that on-farm antibiotic usage is key to selecting the types of E. coli present on these farms. 3GC-R E. coli and 3GC-R plasmids were diverse, suggestive of long-term circulation in this region. We identified the de novo mobilization of the resistance gene blaROB from pig pathogens into E. coli on a novel mobile genetic element, which shows the importance of surveying poorly studied regions for antibiotic resistance that might impact human health.

In vitro synergistic interaction between Melaleuca armillaris essential oil and erythromycin against Staphylococcus aureus isolated from dairy cows.

Staphylococcus aureus frequently causes subclinical mastitis around the world with a high impact on the milk industry and public health. Essential oils (EO) are recognized antimicrobials that can be synergistic with antibiotics. The main objective of this study was to evaluate the essential oil (EO) of Melaleuca armillaris as an adjuvant of erythromycin (ERY) for the alternative treatment of bovine mastitis caused by S. aureus. The Minimum Inhibitory and Bactericidal Concentrations (MIC and MBC) of EO, ERY, and its combinations were established against S. aureus at different pHs (7.4, 6.5 and 5.0), emulating extra and intracellular conditions. Sensitive (N = 3) and resistant (N = 3) strains to ERY and S. aureus ATCC 29213 as control were used. Math models were applied to describe the antibacterial activity of EO and combinations EO-ERY. The EO was bactericidal against all the strains independently of the pH with a slight improvement in acid conditions. The synergism between EO and ERY was estimated by the Fractional Inhibitory Concentration Index (FIC) and by mathematical modeling of the bacterial killing data. Synergism was observed with ERY, where combinations had bactericidal activity also even with pH modification. M. armillaris EO is an interesting adjuvant for ERY, being a promissory option for further analysis of intracellular efficacy against S. aureus.

Melaleuca armillaris Essential Oil as an Antibacterial Agent: The Use of Mesoporous Bioactive Glass Nanoparticles as Drug Carrier.

Bioactive glasses have been proposed for bone tissue engineering due to their excellent biocompatibility and osteo-inductive behaviour. The generation of mesoporous bioactive glass (nano) particles adds a high surface area for the dissolution and release of bioactive ions, and the possibility to load them with different drugs for antibacterial purposes. Essential oils (EO) are an interesting resource for alternative medical therapy, providing antimicrobial compounds that come from organic/natural resources like aromatic plants. Also, a biological polymer, such as chitosan, could be used to control the release of active agents from mesoporous bioactive glass (MBG) loaded particles. This work presents MBG particles with nominal composition (in mol) 60% SiO2, 30% CaO and 10% P2O5, loaded with essential oil of Melaleuca armillaris, which contains 1,8-cineol as the main active component, with an inhibitory in vitro activity against several bacterial species. Also, co-loading with a broad-spectrum antibiotic, namely gentamicin, was investigated. The MBG particles were found to be of around 300nm in diameter and to exhibit highly porous open structure. The release of EO from the particles reached 72% of the initial content after the first 24 h, and 80% at 48 h of immersion in phosphate buffered solution. Also, the MBG particles with EO and EO-gentamicin loading presented in vitro apatite formation after 7 days of immersion in simulated body fluid. The antibacterial tests indicated that the main effect, after 24 h of contact with the bacteria, was reached either for the MBG EO or MBG EO-gentamicin particles against E. coli, while the effect against S. aureus was less marked. The results indicate that MBG particles are highly bioactive with the tested composition and loaded with EO of Melaleuca armillaris. The EO, also combined with gentamicin, acts as an antibacterial agent but with different efficacy depending on the bacteria type.

Modeling the Growth and Death of Staphylococcus aureus against Melaleuca armillaris Essential Oil at Different pH Conditions.

Essential oils (EO) are a great antimicrobial resource against bacterial resistance in public health. Math models are useful in describing the growth, survival, and inactivation of microorganisms against antimicrobials. We evaluated the antimicrobial activity of Melaleuca armillaris EO obtained from plants placed in the province of Buenos Aires (Argentina) against Staphylococcus aureus. The minimum inhibitory and bactericidal concentrations were close and decreased, slightly acidifying the medium from pH 7.4 to 6.5 and 5.0. This result was also evidenced by applying a sigmoid model, where the time and EO concentration necessaries to achieve 50% of the maximum effect decreased when the medium was acidified. Moreover, at pH 7.4, applying the Gompertz model, we found that subinhibitory concentrations of EO decreased the growth rate and the maximum population density and increased the latency period concerning the control. Additionally, we established physicochemical parameters for quality control and standardization of M. armillaris EO. Mathematical modeling allowed us to estimate key parameters in the behavior of S. aureus and Melaleuca armillaris EO at different pH. This is interesting in situations where the pH changes are relevant, such as the control of intracellular infections in public health or the development of preservatives for the food industry.

Pet and Stray Dogs as Reservoirs of Antimicrobial-Resistant Escherichia coli.

The close contact between dogs and humans creates the best bridge for interspecies transmission of antimicrobial-resistant bacteria. The surveillance of its resistance including the detection of extended-spectrum beta-lactamases (ESBLs) in Escherichia coli as indicator bacteria is an important tool to control the use of antimicrobials. The aim of this research was to evaluate the E. coli resistance in strains by phenotypic methods, isolated from pet and stray dogs of La Plata city, Argentina. Faecal samples were collected using rectal swabs from 50 dogs with owners (home dogs = HD) and 50 homeless dogs (stray dogs = SD). They were cultured in 3 MacConkey agar plates, with and without antibiotics (ciprofloxacin and cefotaxime). 197 strains were isolated, of which only 95 strains were biochemically identified as E. coli, 46 strains were from HD, and 49 were from SD. Antimicrobial susceptibility was evaluated by the Kirby-Bauer disk diffusion method. The most prevalent resistance was for tetracycline, streptomycin, and ampicillin. In both groups, the level of resistance to 3rd generation cephalosporins was high, and there were multiresistant strains. There was a higher level of antimicrobial resistance in strains from SD compared to HD. There were 8% of strains suspected of being ESBLs among samples of HD and 36% of SD. One (2%) of the strains isolated from HD and 11 (22%) from SD were phenotypically confirmed as ESBL. Pets and stray dogs are a potential source of E. coli antibiotic resistance in Argentina; therefore, its surveillance must be guaranteed.

Melaleuca armillaris Essential Oil in Combination With Rifaximin Against Staphylococcus aureus Isolated of Dairy Cows.

Staphylococcus aureus is the major subclinical mastitis-causing pathogen in dairy cows. In some European and Latin American countries, rifaximin (RIF) is a commonly used therapy at drying off. Phytotherapeutics are alternatives for the treatment of infectious diseases. Melaleuca armillaris essential oil (EO) has been reported as a good antimicrobial against S. aureus. The aim of this work was to investigate, in vitro, the combined effect of EO and RIF to identify a synergic interaction against S. aureus in order to obtain enough information for subsequent pharmacokinetic/pharmacodynamic studies. The minimum inhibitory concentrations (MIC) for RIF, EO, and combinations of these against S. aureus strains were determined at pH 7.4, 6.5, and 5.0, representing intracellular conditions where S. aureus is usually located. The fractional inhibitory concentration index (FIC) and the index of antibacterial activity (E) were evaluated. The MIC of EO at pH 7.4 was 25-12.5 µL/mL and decreased with the acidity of the medium. RIF presented a high antimicrobial activity (0.032 µg/mL) against S. aureus regardless of the pH conditions. Combining RIF with EO, we found a synergic effect. A mix of 0.004 µg/mL of RIF and 12.5 µL/mL of EO led to a virtual eradication effect against wild-type strains at pH 7.4. Media acidification improves the EO/RIF activity, so EO would be a good adjuvant for RIF to treat staphylococcal infections and decrease antimicrobial resistance.

Combination of Cloxacillin and Essential Oil of Melaleuca armillaris as an Alternative Against Staphylococcus aureus.

The emergence of resistance to antibiotics has been favored by abuse in the application of antimicrobials in human and animal medicine. Essential oils are a great resource to deal with this crisis. Melaleuca armillaris belongs to the family of Myrtaceae, rich in species with essential oils. Plant extracts has shown antimicrobial activity in many investigations. Cloxacillin (CLOX) is an antibiotic widely used in veterinary medicine against Staphylococcus aureus. Our aim was to assess pharmacodynamic interaction established by combining essential oil of M. armillaris (EO) with CLOX in search of a synergistic effect that maximizes the antibacterial activity against S. aureus. The EO was obtained by steam distillation and its composition was analyzed by a GC-FID-MS. The most abundant components in the EO were 1.8 cineole (72.3%), limonene (7.8%). and α-pinene (6%). We worked with wild type S. aureus strains (n = 3) isolated from Holstein cows, and S. aureus ATCC 29213 as the reference strain. The Minimum Inhibitory Concentration (MIC) of CLOX, EO and the combination was determined by microdilution in broth at pH 7.4; 6.5 and 5.0. The checkerboard method was applied to evaluate the interaction between CLOX and EO. The Fractional Inhibitory Concentration index (FIC) was established. From those combinations that yielded the lowest FIC values, we evaluated the index of antibacterial activity (E), established as the difference between the Log10 values of the number of viable bacteria at the initial (nt0) and at the end of the test (nt24). So, time-killing curves with CLOX and EO/CLOX combination at 0.5, 1, 2, 4, and 8 fold the MIC in broth at pH 7.4; 6.5 and 5.0 were prepared. We considered Bacteriostatic effect (E = 0) Bactericidal effect (E = -3) and Effect of virtual eradication of bacteria (E = -4). A clear synergic activity between the EO and the CLOX was demonstrated, which allows reducing the MIC of ß-lactam against S. aureus. This interaction was favored by acidification of the medium, where lower concentrations of CLOX achieved a bactericidal effect, close to virtual eradication, in the presence of small amounts of EO.

Residue depletion of ivermectin in broiler poultry.

Helminth infections are widespread in the poultry industry. There is evidence of extra-label use of some drugs, such as ivermectin (IVM), in broiler poultry. Pharmacokinetic and residual studies of IVM in poultry, however, are rather scarce. Our aim was to determine time restrictions for broiler chickens fed with balanced feed mixed with IVM for 21 days, and thus achieve acceptable residual levels for consumption as established by the European Union. Sixty 1-day-old chicks were fed with food supplemented with IVM at 5 mg kg-1 feed for 21 days. Groups of six treated animals were sacrificed at 0, 1, 2, 4, 8, 10, 15, 20 and 28 days after treatment. Liver, skin/fat, kidney and muscle samples were obtained. IVM were determined by liquid chromatography with fluorescence detection after automatic solid-phase extraction with SPE C18 cartridges. The highest concentrations were measured in the liver, which is logical given that IVM is a drug that undergoes extensive hepatic metabolism. The optimal withdrawal time for edible tissues of these animals to stay within the permitted residual levels were: 12 days for liver, 8 days for skin/fat, 0 days for muscle and 10 days for kidney.