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.

Isolation of lactic acid bacteria from the reproductive tract of mares as potentially beneficial strains to prevent equine endometritis.

Endometritis, the inflammation of the endometrium, is the leading cause of subfertility in mares, and therefore responsible for major economic losses in the horse industry worldwide. It is generally treated with uterine lavages combined with ecbolic agents and local or systemic antibiotics. However, since antibiotic overuse has been associated with antimicrobial resistance in mares with persistent endometritis, new prevention and treatment alternatives are needed. One such alternative could be the use of probiotic lactic acid bacteria (LAB) isolated from the host. Thanks to their species specificity, resident microbiota may restore ecological equilibrium within the host, and therefore, help prevent infections and improve physiological functions. In the present study, 257 bacterial strains were isolated from 77 healthy mares, and 88.76% (n = 228) of them were phenotypically classified as LAB. Within this group, 65.79% were able to inhibit at least one strain from each of the genera that most commonly cause equine endometritis (Streptococcus equi subsp. zooepidemicus, Escherichia coli, and Staphylococcus spp.). Five strains (RCE11, RCE20, RCE91, RCE99, and RCE167) were selected on the basis of their beneficial properties: ability to autoaggregate and adhere to equine epithelial cells, high inhibition of and co-aggregation with all the bacteria isolated from clinical cases of endometritis evaluated, and negative co-inhibition between one another. All five were finally identified as Enterococcus spp., namely E. faecium (two strains), E. hirae (two strains), and E. gallinarum (one strain). Further studies will assess their safety and biotechnological potential for the design of a multi-strain probiotic formula to prevent equine endometritis.

Lactococcus lactis subsp lactis CRL1655 and Schleiferilactobacillus perolens CRL1724 inhibit the adherence of common bovine mastitis pathogens to mammary gland cells, without causing histological changes in the mammary gland.

AIMS: The present work assessed the ability of two selected lactic acid bacteria (LAB) strains (Schleiferilactobacillus perolens CRL1724 and Lactococcus lactis subsp. lactis CRL1655) to inhibit the adherence of bovine mastitis pathogens to mammary epithelial cells (MAC-T) and their effects (if any) on the structure of the gland after intramammary inoculation at dry-off. METHODS AND RESULTS: Established bovine mammary epithelial cells (MAC-T) were used to assess the LAB strains' ability to inhibit the adherence of bovine mastitis pathogens. Monolayers of MAC-T cells were co-cultured with the LABs and then individual pathogen was added. Both strains prevented the adherence of S. aureus RC108, S. chromogenes, S. uberis UT102 and E. coli ATCC 35218. Adherence of the latter two pathogens was inhibited most strongly in vitro. To evaluate the effect of the LAB on the structure of the bovine udders, quarters were intramammary inoculated with the LAB mixture at dry-off. After slaughtering, the teats were dissected and histopathologically analysed. No modifications were identified post-inoculation in the structure of the epithelial, subepithelial and connective tissues of the mammary gland. CONCLUSIONS: Probiotic strains L. lactis subsp lactis CRL1655 and S. perolens CRL1724 were both able to inhibit the adherence of a number of bovine mastitis pathogens in vitro, and that the intramammary inoculation of these strains at the established dose and concentration did not cause significant alterations in the mammary epithelium nor had undesirable effects on tissues, and may therefore be considered harmless. SIGNIFICANCE AND IMPACT OF STUDY: The promising findings demonstrated in this work support the potential of probiotic micro-organisms as a natural and effective alternative to prevent bovine mastitis during the dry-off period.

In Vitro Characterization of Lactic Acid Bacteria Isolated from Bovine Milk as Potential Probiotic Strains to Prevent Bovine Mastitis.

Bovine mastitis causes economic losses on dairy farms worldwide. Lactic acid bacteria (LAB) in animal health are an alternative tool to avoid antibiotic therapy on the prevention of bovine mastitis. In previous studies, 12 LAB isolated from bovine milk were selected taking into account some of the following characteristics: hydrophobicity, auto aggregative capability, inhibition of indicator pathogens, hydrogen peroxide, and capsular polysaccharide production. These LAB were considered because of their beneficial properties. In this work, we also analyzed the antimicrobial activity and the co-aggregation against mastitis causing bacteria, auto-inhibition, adhesion to bovine teat canal epithelial cells (BTCEC), and growth kinetic curves for the 12 LAB. Two of them, Lactococcus lactis subsp. lactis CRL 1655 and Lactobacillus perolens CRL 1724, were selected because they had an interesting pattern of adhesion to BTEC, the inhibition of pathogens and the co-aggregation with the 100% of the assayed pathogens. They showed a predictable difference in the PFGE genomic pattern bands. The kinetic growth of these two strains was similar between them and with the rest of the assayed LAB. The strains selected in the present study showed indispensable characteristics for their inclusion in a probiotic formulation to be used at dry-off period for the prevention of bovine mastitis.

Prevalence and antibiotic susceptibility of coagulase-negative Staphylococcus species from bovine subclinical mastitis in dairy herds in the central region of Argentina.

Coagulase-negative staphylococci (CNS) are a common cause of bovine subclinical mastitis (SCM). The prevalence of CNS species causing SCM identified by genotyping varies among countries. Overall, the antimicrobial resistance in this group of organisms is increasing worldwide; however, little information exists about a CNS species resistant to antibiotics. The aim of the present study was to genotypically characterize CNS at species level and to determine the prevalence and antibiotic resistance profiles of CNS species isolated from bovine SCM in 51 dairy herds located in the central region of the province of Cordoba, Argentina. In this study, we identified 219 CNS isolates at species level by PCR-restriction fragment length polymorphism of the groEL gene. Staphylococcus chromogenes (46.6%) and Staphylococcus haemolyticus (32%) were the most prevalent species. A minimum of three different CNS species were present in 41.2% of the herds. S. chromogenes was isolated from most of the herds (86.3%), whereas S. haemolyticus was isolated from 66.7% of them. The broth microdilution method was used to test in vitro antimicrobial susceptibility. Resistance to a single compound or two related compounds was expressed in 43.8% of the isolates. S. chromogenes and S. haemolyticus showed a very high proportion of isolates resistant to penicillin. Resistance to two or more non-related antimicrobials was found in 30.6% of all CNS. S. haemolyticus exhibited a higher frequency of resistance to two or more non-related antimicrobials than S. chromogenes.

Prevalencia y sensibilidad a antibióticos de especies de estafilococos coagulasa negativos provenientes de mastitis subclínica en bovinos de tambos de la región central de Argentina / Prevalence and antibiotic susceptibility of coagulase-negative Staphylococcus species from bovine subclinical mastitis in dairy herds in the central region of Argentina

Coagulase-negative staphylococci (CNS) are a common cause of bovine subclinical mastitis (SCM). The prevalence of CNS species causing SCM identified by genotyping varies among countries. Overall, the antimicrobial resistance in this group of organisms is increasing worldwide; however, little information exists about a CNS species resistant to antibiotics. The aim of the present study was to genotypically characterize CNS at species level and to determine the prevalence and antibiotic resistance profiles of CNS species isolated from bovine SCM in 51 dairy herds located in the central region of the province of Cordoba, Argentina. In this study, we identified 219 CNS isolates at species level by PCR-restriction fragment length polymorphism of the groEL gene. Staphylococcus chromogenes (46.6%) and Staphylococcus haemolyticus (32%) were the most prevalent species. A minimum of three different CNS species were present in 41.2% of the herds. S. chromogenes was isolated from most of the herds (86.3%), whereas S. haemolyticus was isolated from 66.7% of them. The broth microdilution method was used to test in vitro antimicrobial susceptibility. Resistance to a single compound or two related compounds was expressed in 43.8% of the isolates. S. chromogenes and S. haemolyticus showed a very high proportion of isolates resistant to penicillin. Resistance to two or more non-related antimicrobials was found in 30.6% of all CNS. S. haemolyticus exhibited a higher frequency of resistance to two or more non-related antimicrobials than S. chromogenes.

Los estafilococos coagulasa negativos (ECN) son una causa frecuente de mastitis subclínica (MSC) en bovinos. La prevalencia de especies de ECN causantes de MSC identificadas por métodos genotípicos varía entre países. La resistencia antimicrobiana en este grupo de organismos se está incrementando en el mundo; sin embargo, existe poca información acerca de las especies de ECN resistentes a antibióticos. Los objetivos del presente estudio fueron caracterizar genotípicamente los ECN a nivel de especie y determinar la prevalencia y los perfiles de resistencia a antibióticos de las especies de ECN aisladas de MSC en bovinos de 51 rodeos situados en la provincia de Córdoba, Argentina. Mediante polimorfismos de los fragmentos de restricción del gen groEL identificamos 219 aislamientos de ECN a nivel de especie. Staphylococcus chromogenes (46,6%) y Staphylococcus haemolyticus (32%) fueron las especies más prevalentes. Un mínimo de 3 especies diferentes de ECN estuvieron presentes en el 41,2% de los tambos. S. chromogenes fue aislado en la mayoría de los tambos (86,3%), mientras que S. haemolyticus fue aislado en el 66,7% de aquellos. Para el análisis de sensibilidad a los antimicrobianos in vitro se usó el método de microdilución en caldo. La resistencia a un único compuesto o a 2 compuestos relacionados fue expresada en el 43,8% de los aislamientos. S. chromogenes y S. haemolyticus mostraron una muy elevada proporción de aislamientos resistentes a penicilina. La resistencia a 2 o más antimicrobianos no relacionados fue hallada en el 30,6% de los ECN. S. haemolyticus exhibió una frecuencia de resistencia a 2 o más antimicrobianos no relacionados más elevada que S. chromogenes.

Efficacy of corn silage inoculants on the fermentation quality under farm conditions and their influence on Aspergillus parasitucus, A. flavus and A. fumigatus determined by q-PCR.

Laboratory-scale silos were prepared to evaluate the efficacy of two different lactic acid bacteria (LAB) on the fermentation quality and mycobiota of corn silage. Their influence on Aspergillus species' variability by using the q-PCR technique was studied. Silage inoculated with Lactobacillus rhamnosus RC007 or L. plantarum RC009 were compared with uninoculated silage. Silos were opened after 1, 7, 45, 90 and 120 days after ensiling. At the end of the ensiling period, silos were left open for 7 days to evaluate aerobic stability. Rapid lactic acid production and decline in pH values were seen in the early stages of fermentation in silage inoculated with L. rhamnosus RC007. After aerobic exposure, a significant decline in lactic acid content was observed in untreated and L. plantarum RC009-inoculated silages. Counts for yeasted and toxigenic fungus remained lower, after aerobic exposure, in L. rhamnosus RC007-inoculated silage, in comparison with L. plantarum RC009 and uninoculated silages. Comparing the influence exerted by both BAL, it was observed that L. rhamnosus RC007 was more efficient at inhibiting the three fungal species tested whose DNA concentrations, determined by q-PCR, oscillated near the initial value (pre-ensiling maize). The ability of L. rhamnosus RC007 to produce lactic acid rapidly and the decline in pH values in the early stages of the fermentation along with the reduction of yeast and mycotoxicogenic fungus after aerobic exposure shows its potential as a bio-control inoculant agent in animal feed.

Histological examination of non-lactating bovine udders inoculated with Lactobacillus perolens CRL 1724.

The effect of intramammary inoculation of Lactobacillus perolens CRL 1724 on bovine udders at drying off was evaluated through histological examination of the canal and cistern tissues. The persistence of the strain in the udder 7 d post inoculation was also determined. Lb. perolens CRL 1724 was recovered from all mammary quarters and no clinical signs or teat damage were observed after inoculation of 10(6) cfu/ml. The udders showed a normal structural aspect and there were no modifications of the milk appearance. Lb. perolens CRL 1724 cells were evidenced on the surface of the epithelial cells of the cistern without causing any morphological modifications or cell alterations. Lb. perolens CRL 1724 produces a mild inflammatory reaction, characterized by recruitment of neutrophils to the epithelial zone and a slight hyperaemia into blood vessels. This preliminary study provides important information for further studies directed towards the inclusion of Lb. perolens CRL 1724 in the design of probiotic products for preventing bovine mastitis in non-lactating dairy cows.

Lactic acid bacteria from raw milk as potentially beneficial strains to prevent bovine mastitis.

Bovine mastitis produces a wide variety of problems in the dairy farm. The treatment of this disease is based on the use of antibiotics which are not always effective. These drugs are also responsible for the presence of residues in the milk and the increase of antibiotic-resistant strains. Probiotic products were proposed as a valid alternative to antibiotic therapies and are also useful for the prevention of infectious syndromes. With the aim of designing a probiotic product to prevent bovine mastitis, lactic acid bacteria (LAB) were isolated from foremilk samples from different dairy farms in Córdoba-Argentina. One hundred and seventeen LAB were isolated and their beneficial characteristics such as the production of inhibitory substances, surface properties and production of exopolysaccharides (EPS) were assessed. Most of them displayed low degree of hydrophobicity, autoaggregation, EPS negative phenotype and were identified as Enterococcus hirae and Pediococcus pentosaceus. Nine LAB strains inhibited three indicator bacteria. Some isolates were pre-selected and genetically identified according to the results obtained. Antibiotic resistance and virulence factors were studied for the assessment of the safety of the strains. The results obtained were compared to those reported previously from samples obtained in the North-western area of the country and some differences were found.

Effects of intramammary inoculation of Lactobacillus perolens CRL1724 in lactating cows' udders.

Bovine mastitis is the most important infectious disease on dairy farms. Conventional antibiotic therapy is often unsatisfactory and alternative treatments are continually under investigation. Lactobacillus (Lb.) perolens CRL 1724 and Lactobacillus plantarum CRL 1716 were previously isolated from milk of dairy cows and selected according to their potential probiotic properties. In the present work the in-vitro capacity of Lactobacillus strains to adhere to bovine teat canal epithelial cells (BTCEC) and to inhibit and co-aggregate 14 mastitis-causing pathogens (MCPs) was investigated. The effect of Lb. perolens CRL 1724 after intramammary inoculation in lactating cows was evaluated through determination of clinical signs of mastitis, milk appearance, somatic cell counts and Lb. perolens CRL 1724 recovery from milk. Lb. perolens CRL 1724 was able to inhibit 12 of 14 MCPs (85·7%) in vitro, especially those considered to be major pathogens. In addition, Lb. perolens CRL 1724 co-aggregated with all of them. Lb. plantarum CRL 1716 was able to inhibit 7 of 14 MCPs (50%) in vitro and showed co-aggregation ability similar to Lb. perolens CRL 1724. Lb. perolens CRL 1724 showed a higher efficacy of adhesion to BTCEC (values of percentage of adhesion and adhesion index of 75% and 14·4, respectively) than Lb. plantarum CRL 1716 (37% and 7·4, respectively). Lb. perolens CRL 1724 was recovered from all mammary quarters and no clinical signs or teat damage were observed after the inoculation of 106 cfu/ml. The udders presented a normal aspect and there were no changes in the appearance of the milk. The results obtained will serve as the basis for further trials to evaluate the potential of Lb. perolens CRL 1724 to be included in a non-antibiotic formulation for the prevention of bovine mastitis.

Efficacy of immunization against bovine mastitis using a Staphylococcus aureus avirulent mutant vaccine.

Bovine mastitis is responsible of major economic losses on dairy farms worldwide. In Argentine dairy herds, Staphylococcus aureus is the main causative agent of the disease. The ineffectiveness of some current practices to control S. aureus infections, often leads to a chronic and recurrent infection with persistent bacterial reservoir within a herd. Vaccination against S. aureus seems to be a rational approach for the control of the disease. In the present study, we investigate the response of dairy heifers after a combined immunization schedule with the avirulent mutant RC122 S. aureus vaccine. Vaccinated and non-vaccinated heifers were challenged 40 days after calving with the parental virulent strain. After challenge, and during the study period, milk bacterial recovery was significantly higher in non-vaccinated heifers than vaccinates. Importantly, inoculated bacteria could not be isolated from the milk of vaccinated heifers until 72 h after challenge, and the overall percentage of infected quarters in these animals was significantly lower. An increase in the level of specific IgG was observed in blood and milk of vaccinated heifers during the trial. At calving, IgG(2) was the main antibody isotype found in blood. Immune sera from vaccinated heifers increased phagocytosis over sera from non-vaccinated heifers and were able to opsonize heterologous S. aureus strains. Results demonstrated that immunization of dairy heifers with strain RC122 was able to elicit a significant opsonic antibody production in blood and milk and provides protection by a significant reduction in bacterial shedding after challenge.

Experimental trial in heifers vaccinated with Staphylococcus aureus avirulent mutant against bovine mastitis.

Staphylococcus aureus, is the most frequently isolated pathogen from cases of bovine mastitis. Vaccination against S. aureus seems to be a rational approach for the control of staphylococcal mastitis. In the present work we evaluate the response of heifers vaccinated with a S. aureus avirulent mutant to the intramammary challenge with a S. aureus virulent strain. Clinical signs, production of milk, shedding of S. aureus cells, somatic cell count (SCC) and antigen-specific IgG in blood and milk, were determined. Two subcutaneous doses of a culture of the mutant, used as vaccine, was administered to four pregnant heifers 30 and 10 days before calving. The vaccinated heifers and four non-vaccinated were challenged 10 days after calving with the homologous virulent S. aureus strain, which was inoculated by intramammary route into two quarters of each animal. No local tissue damage was observed due to the administration of the vaccine. A significantly increase of specific IgG to S. aureus RC122 was detected in blood and milk of vaccinate heifers as well as a slight increase in daily milk yield during the trial. No significant difference on shedding of bacteria in milk and SCC were found among groups. In conclusion, vaccination of heifers before calving by an avirulent mutant vaccine of S. aureus, induced specific and significant antibody responses and provide better post-challenge conditions in vaccinated heifers.