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.

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.

Shiga toxin-producing Escherichia coli in beef retail markets from Argentina.

Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that cause mild or serious diseases and can lead to people death. This study reports the prevalence and characteristics of STEC O157 and non-O157 in commercial ground beef and environmental samples, including meat table, knife, meat mincing machine, and manipulator hands (n = 450) obtained from 90 retail markets over a nine-month period. The STEC isolates were serotyped and virulence genes as stx (Shiga toxin), rfb(O157)] (O157 lipopolysaccharide), fliC(H7) (H7 flagellin), eae (intimin), ehxA (enterohemolysin) and saa (STEC autoagglutinating adhesin), were determined. STEC O157 were identified in 23 (25.5%) beef samples and 16 (4.4%) environmental samples, while STEC non-O157 were present in 47 (52.2%) and 182 (50.5%), respectively. Among 54 strains isolated, 17 were STEC O157:H7 and 37 were STEC non-O157. The prevalent genotype for O157 was stx(2)/eae/ehxA/fliC(H7) (83.4%), and for STEC non-O157 the most frequent ones were stx(1)/stx(2)/saa/ehxA (29.7%); stx(2) (29.7%); and stx(2)/saa/ehxA (27%). None of the STEC non-O157 strains were eae-positive. Besides O157:H7, other 20 different serotypes were identified, being O8:H19, O178:H19, and O174:H28 the prevalent. Strains belonging to the same serotype could be isolated from different sources of the same retail market. Also, the same serotype could be detected in different stores. In conclusion, screening techniques are increasingly sensitive, but the isolation of STEC non-O157 is still a challenge. Moreover, with the results obtained from the present work, although more studies are needed, cross-contamination between meat and the environment could be suspected.

Efecto del Ácido Clorhídrico y Ácido Láctico sobre el desarrollo de treinta cepas de Listeria spp. aisladas de alimentos / Effect of Chlorhidric and Lactic Acid on the development of thirty strains of Listeria spp. from foods stuffs

Listeria monocytogenes es el agente causal de Listeriosis en humanos, siendo los alimentos “listos para comer” una de las principales vías de transmisión. El pH óptimo de desarrollo de L. monocytogenes es entre 6 y 9, tolerando hasta pH 4,4. El objetivo fue estudiar el comportamiento in vitro de 30 cepas de Listeria spp. aisladas de alimentos, modificando el pH por adición de HCl y ácido láctico. Se utilizó caldo cerebro corazón al que se le agregó HCl ó ácido láctico hasta pH de 4,8, 5,2, 5,5, 5,8 y 6. Con HCl a pH 4,8 se desarrolló una cepa de L. monocytogenes tipo 1, a pH 5,5 se desarrollaron el 50% de las cepas. Con ácido láctico, a pH 4,8 no hubo desarrollo. A pH 5,8 y 6 con ambos ácidos se desarrollaron la mayoría de las Listerias analizadas. El ácido láctico presentó mayor efecto inhibidor que HCl.

Listeria monocytogenes is the causative agent of listeriosis in humans, being the “ready to eat” foods one of the main transmission ways. The optimun pH for L. monocytogenes to grow was between 6 and 9, tolerating up to pH 4.4. The objetive was to study the in vitro behavior of thirty strains Listeria spp. isolated from foods, modifying the pH adding HCl and lactic acid. Brain heart broth added with HCl or lactic acid until pH 4.8, 5.2, 5.5, 5.8 and 6 was used. With HCl at pH 4.8 one strain of L. monocytogenes type 1 developed. At pH 5.5, 50% of the strains developed. With lactic acid, at pH 4.8 growth was not observed. At pH 5.8 and 6 with both acids grew most of the Listeria analyzed. The lactic acid had a greater inhibitory effect than HCl.